- Lindsay Lohan as Cady Heron, a 16-year-old girl who transfers to a public high school after being homeschooled her whole life in Africa
- Jessie Wright plays Cady at age 5
- Rachel McAdams as Regina George, a rich, popular mean girl and the leader of The Plastics
- Tina Fey as Ms. Norbury, math teacher and Mathletes moderator
- Tim Meadows as Mr. Duvall, the principal of North Shore High School
- Amy Poehler as Mrs. George, Regina's mother
- Ana Gasteyer as Mrs. Heron, Cady's mother
- Lacey Chabert as Gretchen Wieners, a rich Jewish girl and member of the Plastics, Regina's second-in-command
- Lizzy Caplan as Janis Ian, Cady and Damian's goth friend and artist
- Daniel Franzese as Damian, Cady and Janis' openly gay friend
- Neil Flynn as Mr. Heron, Cady's father
- Jonathan Bennett as Aaron Samuels, Regina's ex-boyfriend and Cady's love interest
- Amanda Seyfried as Karen Smith, a dimwitted member of the Plastics
- Rajiv Surendra as Kevin Gnapoor, the Mathletes president and an amateur rapper who is attracted to Janis
The film's large cast also includes Molly Shanahan and Jonathan Malen as Kristen Hadley and her boyfriend, Ky Pham and Danielle Nguyen as "Cool Asian" students Trang Pak and Sun Jin Dinh, and Daniel DeSanto and Diego Klattenhoff as jocks Jason and Shane Oman, Gretchen and Regina's respective boyfriends. Dwayne Hill plays predatory gym teacher Coach Carr.
Alisha Morrison plays "unfriendly" student Lea Edwards and Jan Caruana plays overweight student Emma Gerber. Wai Choi plays mathlete Tim Pak. Julia Chantrey plays Amber D'Alessio and Jacky Chamberlain and Olympia Lukis appear as disabled students Giselle Sgro and Jessica Lopez. Stefanie Drummond plays offbeat student Bethany Byrd.
Tyson Fennell appears as Cady's childhood crush Nfume. Alexandra Stapley portrays student Taylor Wedell and Laura DeCarteret plays her mother, while Nicole Crimi portrays Regina's younger sister Kylie and Dan Willmott appears as her father. Sharron Matthews portrays Mr. Duvall's secretary Joan and Clare Preuss plays Marymount Prep student Caroline Krafft.
