Mary Jane first appears as Peter Parker narrates the first few minutes of the film. She is depicted as a popular girl at Midtown High School and the girlfriend of star athlete Flash Thompson, while Peter, her next-door neighbor who has had a crush on her since they were in first grade, is an academically gifted but shy outsider. Peter struggles to catch the school bus until MJ asks the driver to stop for him. Later, their class attends a field trip to a lab at Columbia University. Peter's friend Harry Osborn flirts with MJ before Peter takes a picture of her for the school newspaper, after which a genetically-altered spider bites him, ultimately giving him his abilities. Peter then saves Mary Jane from slipping in the school cafeteria with his new abilities the next day and inadvertently triggers a fight with Flash, though he is able to easily dodge Flash's punches and knocks him out with one punch. Peter later apologizes to MJ for the fight while she confides in him about wanting to escape her abusive, alcoholic father and pursue an acting career.

She breaks up with Flash during high school graduation and takes up work as a waitress while looking for acting gigs. While Mary Jane starts dating Harry, she and Peter begin to form a closer friendship. Peter rescues her as Spider-Man when the Green Goblin attacks a parade she was attending with Harry, and again later on from a gang in an alley. Mary Jane thanks Spider-Man with a kiss without him revealing his identity. MJ grows distant from Harry due to his desire to please his father, Norman, at her expense and also subsequently develops feelings for Peter when she realizes how much he cares for her, causing Harry to break up with MJ upon seeing her and Peter holding hands.

Norman, who is the Green Goblin and has deduced Peter is Spider-Man, kidnaps Mary Jane upon realizing Peter loves her, thanks to his son's unwitting confession. The Goblin forces Peter to choose between saving Mary Jane and a Roosevelt Island Tramway car full of children, all of whom he holds hostage on top of the Queensboro Bridge. Spider-Man opts to save both, and despite a struggle, manages to lower MJ and the kids onto a boat, after which the Goblin whisks Spider-Man away for a final confrontation which results in the Goblin's death. At Norman's funeral, Mary Jane tells Peter that she loves him and they kiss. Peter, who decides that for her protection they can't be together, gently rejects her and insists they can only be friends. As he leaves, a saddened Mary Jane realizes that her kiss with Peter reminds her of the one she shared with Spider-Man and suspects that he is the superhero.
Two years after the events of the first movie, Mary Jane has found success as a model and Off-Broadway actress. Maintaining her friendships with both Peter and Harry, she continues to pine for Peter, but as he continues to reject her, she starts dating astronaut John Jameson, the son of Peter's boss J. Jonah Jameson. Struggling to balance his vigilantism as Spider-Man with his personal life, Peter fails to make it to one of Mary Jane's performances of The Importance of Being Earnest.

Fed up with being Spider-Man and after losing his powers, Peter tosses his suit away and is able to handle his job and studies and decides to pursue MJ, though she pushes him away when John proposes to her. After Mary Jane kisses John, in a manner that is reminiscent of the upside-down kiss between her and Spider-Man from the first film, she realizes that she does not love Joh…

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• Mary Jane also appears in Spider-Man: The New Animated Series, an animated television series serving as a loose alternate continuation of the first Spider-Man film. She, Peter and Harry attend Empire State University together, and she attempts to start a relationship with Peter without much success, owing to his reluctance to endanger her due to his duties as Spider-Man. Mary Jane is voiced by Lisa Loeb.
• Dunst's Mary Jane Watson appears through archive material from Spider-Man in the Into the Spider-Verse sequel Spider-Man: Across the Spider-Verse (2023).
• Mary Jane Watson appears in all three video game adaptations of the films. While she is voiced by Cat O'Conner in the first game, Kirsten Dunst reprises her role in a vocal capacity in the second game while Kari Wahlgren takes over the role in the third game.

Kirsten Dunst's portrayal of Mary Jane Watson in the Spider-Man films was generally well-received, as she was nominated for several awards for her performance, winning four, including the Empire Award for Best Actress in 2003. Owen Gleiberman of Entertainment Weekly remarked on Dunst's ability to "lend even the smallest line a tickle of flirtatious music". In the Los Angeles Times review, critic Kenneth Turan noted that Dunst and Maguire made a real connection on screen, concluding that their relationship "involved audiences to an extent rarely seen in films".

However, elements of her character were met with a mixed response. In his review of Spider-Man 3, Ryan Gilbey of the NewStatesman was critical of Dunst's character: "the film-makers couldn't come up with much for Mary Jane to do other than scream a lot". Allie Gemmill of Bustle wrote in 2017 that the films portray Mary Jane as a constant damsel in distress and gave the illusion that she was defined primarily through the men in her life. Conversely, Adadora Asidianya wrote that Mary Jane provides a voice of reason for Peter, whether as a friend or a love interest.

Spider-Man and Mary Jane's "upside-down kiss" in Spider-Man is now regarded as one of the most iconic moments in film history. Entertainment Weekly put "the kiss in Spider-Man" on its end-of-the-decade "best-of" list, saying: "There's a fine line between romantic and corny. And the rain-soaked smooch between Spider-Man and Mary Jane from 2002 tap-dances right on that line. The reason it works? Even if she suspects he's Peter Parker, she doesn't try to find out. And that's sexy."