Sixteen-year-old homeschooled Cady Heron (Lindsay Lohan) and her zoologist parents (Ana Gasteyer and Neil Flynn) return to the United States after a 12-year research trip in Africa, settling in Evanston, Illinois. Cady attends a public school for the first time, meeting new classmates Janis (Lizzy Caplan) and Damien (Daniel Franzese). The two warn Cady to avoid the school's most exclusive clique, the Plastics, who are led by queen bee Regina George (Rachel McAdams). The Plastics take an interest in Cady, however, and start to invite her to sit with them at lunch. Seeing that Cady is slowly becoming one of The Plastics, Janis hatches a plan of revenge against Regina, using Cady as the infiltrator.
Cady soon learns about Regina's "Burn Book", a notebook filled with rumors, secrets, and gossip about the other girls and some teachers. Cady also falls in love with Regina's ex-boyfriend, Aaron Samuels (Jonathan Bennett), whom a jealous Regina steals back at a Halloween party. Cady continues with Janis's plan to cut off Regina's "resources", which involve separating her from Aaron; tricking her into eating nutrition bars that make her gain weight; and turning Regina's fellow Plastics – insecure rich girl Gretchen Wieners (Lacey Chabert) and sweet but ditzy Karen Smith (Amanda Seyfried) – against her. In the process, Cady unwittingly remakes herself in Regina's image, becoming spiteful, superficial, and abandons Janis and Damian.
When Regina is finally made aware of Cady's treachery, she responds by spreading around the contents of her Burn Book, quickly inciting a riot. To avoid suspicion, Regina inserts a fake libel of herself in the book in order to blame the only female students not mentioned in the book, The Plastics. Principal Ron Duvall (Tim Meadows) soon quells the riot, and ends up sending all the girls in the school to gather in the auditorium. Math teacher Ms. Sharon Norbury (Tina Fey), whom the Burn Book slandered as a drug dealer, makes the girls mentioned in the book fess up to the rumors and apologize to the other students and teachers. When Janis's turn comes, she confesses her plan to destroy Regina with Cady's help and openly mocks Regina with the support of the entire school. Pursued by an apologetic Cady, Regina storms out and gets hit by a school bus, breaking her spine.
Without any friends, shunned by Aaron, and distrusted by everyone, Cady takes full blame for the Burn Book. Her guilt soon dissolves and she returns to her old personality. As part of her punishment for lying and failing Norbury's class, she joins the Mathletes in their competition. There, while competing against an unattractive girl, Cady realizes that mocking the girl's appearance would not stop the girl from beating her. She then realizes that the best thing to do is just solve the problem in front of you and ends up winning the competition after her opponent answers incorrectly. At the Spring Fling dance, Cady is elected Queen, but declares that all her classmates are wonderful in their own way, whereupon she breaks her plastic tiara and distributes the pieces. Cady makes amends with Janis and Damian, reconciles with Aaron, and reaches a truce with the Plastics, who then disband.
By the start of the new school year, the fate of the newly-disbanded Plastics are shown. Regina joins the lacrosse team, Karen becomes the school weather reporter, while Gretchen joins and befriends the "Cool Asians." Aaron graduates from high school and attends Northwestern University, Janis and Kevin Gnapoor begin dating, and Cady declares that she is now normal. Regina walks past Cady and smiles, showing that they are now kind to one another. As this happens, Damien points to Cady the new "Junior Plastics" walking by, and Cady imagines the Junior Plastics being hit by a bus.