Cady Heron has been homeschooled while living in Africa with her zoologist parents for 12 years. Now in Evanston, Illinois, she is entering public high school for the first time. On her first day, she meets fellow students Janis and Damian, who give her a brief "rundown" of the school's cliques, pointing out the students sitting at each of the different cafeteria tables at lunch. After a fellow student named Jason tries using innuendo to flirt with Cady, Regina George (the most popular girl in school) embarrasses him into retreating. Regina and her sidekicks Karen Smith and Gretchen Weiners, known as The Plastics, admire Cady for her innocence and good looks. They invite her to have lunch with them under a long list of conditions: no wearing a ponytail more than once a week, only wearing pink on Wednesdays, etc. Janis, who considers Regina her sworn mortal enemy, devises a plan for Cady to infiltrate The Plastics and exact revenge.
Cady develops a crush on a fellow math student, Aaron Samuels, but Gretchen explains to her that he is Regina's ex and thus completely off-limits. Cady's interest in him prompts Regina to rekindle their relationship directly in front of Cady at a Halloween party, causing Cady to run off to Janis and Damian and jumpstart their plan. Although the math teacher Ms. Norbury encourages her to join the Mathletes, Cady pretends not to understand math so that she can ask Aaron to tutor her, and he agrees. When Cady tells Aaron that Regina was unfaithful to him, he and Regina break up again. Continuing her sabotage, Cady tricks Regina into eating weight-gain bars, under the guise of weight loss bars. She also masterminds a feud between Gretchen and Regina by making Gretchen feel insecure about not receiving a signed candy cane note from Regina, which reaches a breaking point at the Winter Talent Show.
Despite claiming only to be "infiltrating" The Plastics for Janis and Damian, Cady begins to resemble them more and more, eventually replacing not only Gretchen but Regina, becoming the leader of the group. Janis and Damian renounce her friendship. After Regina learns about the weight gain bars, she retrieves a Burn Book in her bedroom, full of harmful gossip and vicious insults about the various students and faculty members at North Shore High, including a couple contributed by Cady herself. After writing slanderous comments about herself, Regina turns the book in to the principal, claiming it to be the work of Cady, Gretchen, and Karen, and then litters the school with photocopies of its pages. The pages cause a riot that makes the principal call an assembly in the gymnasium, where Ms. Norbury lectures the girls and makes them write apologies and perform trust falls. After Janis's apology reveals the full extent of her and Cady's plan to sabotage The Plastics, Regina storms out and is struck by an oncoming school bus.
Rumors fly that Cady pushed Regina in front of the bus, and everyone including Janis and Damian ostracize her. Her parents register their disapproval of her behavior as well, and her father tries grounding her. One day in math class, Cady confesses to having written in the Burn Book, and accepts blame for it. As a punishment, Ms. Norbury has Cady join the Mathletes so that she can participate in the championship tournament. While facing off against her opponent in a "sudden death" round, Cady realizes that only genuine hard work will solve her problem and lead to success, rather than petty insults or emotional manipulation. She is able simultaneously to provide the right answer, and to leave behind the range of toxic behaviors instilled in her by The Plastics.
Encouraged by her Mathletes peers and Ms. Norbury to attend the Spring Fling dance even though she is grounded, Cady goes and finds that she has been voted Spring Fling Queen. In her victory speech, Cady renounces the title and shatters the tiara into pieces, distributing it to her classmates as a symbol of their equality. By the beginning of the new school year, much has changed: the Plastics have disbanded; Regina has joined the lacrosse team; Gretchen has somehow joined the "cool Asians" clique; and Karen is now the school's weather-girl. Cady returns to being friends with Janis and Damien, who have forgiven her, and the three of them remain on the lookout for upcoming "junior Plastics."