Charlie (played by Felicia Day) first crosses paths with Sam and Dean when working as an I.T. expert at Richard Roman Enterprises. She is initially reluctant to get involved in the supernatural world by helping the Winchesters, but becomes a more reliable ally in her second episode and has decided to become a hunter herself by her fourth episode. She quickly a friend and even a surrogate little sister to the Winchesters. By the end of "The Girl with the Dungeons and Dragons Tattoo", it is revealed that her real name is not Charlie Bradbury but merely one of her aliases, and that she has had to go into hiding before. "Pac-Man Fever" delves into her past and it is indicated that "Charlie's" true surname is Middleton. "There's No Place Like Home" reveals that Charlie's given name is in fact Celeste Middleton. She is the only prominent LGBT character on the show; in her first episode, she informs the brothers she is lesbian when she is asked to flirt with a male guard to gain access to a restricted area.

Charlie's first appearance is in the season seven episode "The Girl with the Dungeons and Dragons Tattoo", where Dick Roman asks her to decrypt Frank Devereaux's hard drive. Once she succeeds, she starts reading files describing the Leviathans and their activities, including their connection to Dick Roman. Although she doesn't believe it at first, she realizes the truth when she witnesses a leviathan eat her supervisor and shapeshift to replace him—something Dick explains they cannot do to Charlie, as she possesses a rare "spark" that can't be perfectly replicated. Sam and Dean recruit a terrified Charlie to retrieve Frank's hard drive to protect the information there, as well as to break into Dick's office and hack his emails to gain information for their side. She does so, which alerts Sam and Dean to a secret package meant for Dick which they then steal (the package is later revealed to hold the secret to defeating the leviathan). She is nearly killed when Dick realizes her duplicity, but is saved by Bobby's ghost holding the leviathan off—breaking Charlie's arm in the process—whilst Sam and Dean arrive and rescue her. She leaves after telling Sam and Dean not to contact her again.

Charlie reappears in the season eight episode "LARP and the Real Girl". Now under a different alias, she is the queen of one of four kingdoms of the LARPing game of Moondoor. When two of her "subjects" are killed, it draws Sam and Dean's attention and they head to Moondoor. Charlie's initial reaction is to flee and start a new life again, fearing that she will once more become a target for monsters. After learning why Sam and Dean are there, she agrees to help them investigate the deaths and other mysterious injuries that have happened. Charlie is eventually captured by a good fairy named Gilda who has kidnapped her on her master's orders. Charlie is instantly smitten with Gilda. Dean, Sam and Gerry eventually find Charlie with help from the "prisoner", only to find that she is making out with Gilda. It is revealed that Gerry is Gilda's master; he has unrequited feelings for Charlie and has bought a spellbook with which to control Gilda and force her to kill other players as part of a scheme to make Charlie fall in love with him. Charlie eventually destroys the book to set Gilda free and render Gerry powerless. Afterward, she and Gilda share a goodbye kiss before Gilda returns to her world with Gerry to have Gerry punished. Charlie decides to stop running and changing her identity and to stay and make a life for once. She tells Sam and Dean to call her if they ever need her help again. The three of them participate in a mock battle between their group and the other LARPers, which Charlie's side wins. Charlie's backstory is explored when she returns in "Pac-Man Fever" to give Sam and Dean a case. After she proves to be an excellent shot, Dean takes her as his (temporary) partner instead of Sam, who has become increasingly ill from the trials. Charlie is eventually captured by the creature they are hunting, a type of djinn, who poisons Charlie and places her in a nightmare from which she won't wake up from. In her nightmare, she is trapped in a video game she stole as a kid, recreated, and gave out for free, and where she must endlessly protect her hospitalized mother from super-soldier vampires. It is revealed that her parents had been involved in a car accident when driving over to pick Charlie up from a sleepover, resulting in her father dying and her mother rendered brain-dead. Charlie has been paying for the care of her mother, whom she refuses to let go due to her feelings of guilt over the accident. When Dean takes a potion to enter her nightmare himself to save her, he convinces Charlie that she must let her mother go in order to move on. She then wakes up and in the episode's last scene, takes Dean's advice by having her mother taken off of life-support after reading The Hobbit aloud to her like her mother did to Charlie as a little girl.

Charlie is called in by the Winchesters for help in the ninth season episode "Slumber Party" in order to reconfigure an ancient computer at the Men of Letters' bunker. A childhood fan of L. Frank Baum's Oz books, Charlie is excited when the real Dorothy is found in the bunker. The group works together to try to find a way to defeat The Wicked Witch of the West. Charlie dies to protect Dean from the Witch, but he has Gadreel bring her back to life and tells her that she had only been knocked out, though she learns the truth anyway from Dorothy; Charlie later agrees to keep her death and subsequent resurrection a secret from Sam so long as Dean agrees to one day explain what had really happened. Charlie and Dorothy eventually devise a way to kill the Witch, but are attacked by Sam and Dean, who are both possessed by the Witch. While Dorothy holds the brothers off, Charlie finds and kills the Witch by stabbing in the head with Oz's famous ruby slippers before the Witch can let her army in through a portal to Oz. When Dorothy extends an offer to Charlie to come with her to Oz, Charlie jumps at the prospect of finally getting the kind of magic and adventure she wants. After bidding their goodbyes to the brothers, Charlie and Dorothy cross over to Oz, leaving Sam and Dean to speculate if and when they will come back.

In the season 10 episode "There's No Place Like Home", Sam and Dean find out that Charlie is back from Oz and is attacking people who helped cover up her parents deaths. To their surprise, they find there are two Charlie's, a good and a dark Charlie. The good Charlie explains that she and Dorothy fought a war in Oz to free it from evil and in order to win, Charlie made a deal with the Wizard of Oz to split herself into her good and dark sides. Charlie's dark side single-handedly won the war and Dorothy and the Wizard now lead Oz. Now, dark Charlie has come to Earth seeking revenge for her parents deaths, good Charlie following to stop her. As dark Charlie broke the Key to Oz so Charlie couldn't return, she and Sam investigate the Key in hopes of finding a way to fix it, and eventually track down a former Man of Letters named Clive Dylan who was trapped in Oz after discovering the Key. Dean meanwhile tries to protect Russell Wellington, the man who killed Charlie's parents, but Dark Charlie tricks Dean, kills Russell, and follows Dean to Clive's house where they fight and he brutally beats her, hurting Good Charlie who is still connected to her. At the same time, Sam and Charlie learn that the Wizard is in fact Clive's dark side, split from him as Charlie's was. Unable to fix the Key and to save Oz, Clive shoots himself to force the Wizard to Earth to heal Clive and save himself. Unable to defeat the Wizard, Clive signals Charlie to shoot him which she hesitantly does, killing Clive and the Wizard. Sam stops Dean from beating Dark Charlie to death and they are able to reverse the Wizard's spell and reunite the two Charlie's. Afterwards, Charlie, unable to return to Oz, decides to dedicate herself to finding a way to save Dean from the Mark of Cain and forgives his actions.

Charlie returns to the brothers several episodes later, having found an ancient book known as the Book of the Damned, which is said to document every curse as well as their cures. Upon acquiring the book, a group of unknown men, all with the same tattoo on their wrist, begin to pursue her. After a violent encounter with them, Charlie is shot but narrowly escapes, and calls the Winchesters from a telephone booth, informing them of her discovery and injury, mentioning she sewed it back together with dental floss, a testament to her resourcefulness. When reunited, Sam and Charlie begin their attempt to decipher the book's text, as it is written in an unknown ancient language, but Dean insists they stop, and destroy the book entirely, worrying that the Mark will cause him to use the its information to do evil. Sam and Charlie decide not to destroy it but to put it away in a curse box, which will prevent anyone from tracking it down. Meanwhile, Dean goes to find Charlie's pursuers, who Sam had determined to be members of the historically-prominent Styne family. When they attack the house Charlie and the Winchesters had been staying at, Dean instructs Sam to destroy the book before the men can take it. Sam throws the book in the fire, and the trio defeats and kills their attackers. However, it is later revealed he threw another book in, and hid the real book in hopes he could still cure Dean, despite his brother's wishes. When Charlie, Sam and Dean return to the bunker, they are greeted by Castiel, and Charlie is excited to meet him for the first time. Upon hugging him, she comments she had expected him to be shorter. At Charlie's joking request, Castiel heals her carpal tunnel and even her bullet wound without being asked, having gotten his full power back. The four then have a dinner, laughing and enjoying each other's company.

During "Dark Dynasty", Charlie is called in by Sam to work with Rowena on how to translate the Book of the Damned. Charlie is stunned that Sam lied about destroying the Book and reluctantly agrees to work on translating the Book behind Dean's back. However, Charlie is forced to work with Rowena who annoys her greatly with her disgust at Charlie being a nerd instead of a witch and her insistence that they are similar. Unable to take it anymore, Charlie asks Castiel to let her go cool off for awhile and slips out while he ties up Rowena in another room. Going to a motel, Charlie continues her efforts to translate the Book of the Damned and finally manages to crack the code just before Eldon Styne arrives. Charlie contacts Sam and Dean who tell her to give him the Book which she doesn't have or her notes to save her life. However, unwilling to betray her friends or give the Styne Family the power of the Book, Charlie smashes her iPad and is killed by Eldon. A devastated Sam and Dean find her body soon afterwards. However, unknown to them, Charlie's last act was to email Sam her notes on how to translate the Book, giving them the chance to finish what she started.