During a benefit concert in Paris, France, David Seville (Jason Lee) is injured. Having to recuperate there, Dave asks his aunt (Kathryn Joosten) to look after the Chipmunks, Alvin (Justin Long), Simon (Matthew Gray Gubler) and Theodore (Jesse McCartney). Arrangements are also made for them to go to school at West Eastman High School. After another accident, the Chipmunks are left in the care of Toby (Zachary Levi), Jackie's grandson. Meanwhile, Ian Hawke (David Cross) lives in the basement of JETT Records. Three singing female chipmunks, Brittany (Christina Applegate), Jeanette (Anna Faris) and Eleanor (Amy Poehler), also known as The Chipettes, emerge and Ian hires them as plot to get back at the Chipmunks and revive his career.
While at school, the Chipmunks are bullied by jocks. They visit the principal's office, only to discover that the principal, Dr. Rubin (Wendie Malick) is a huge fan and wants them to help raise money for a music program by participating in a contest. Meanwhile, Ian is shocked to find the Chipmunks on the front page of his newspaper. After he reads a story about them, he quickly sends the Chipettes to school.
When the Chipmunks meet the Chipettes, a rivalry forms after the former group learns that the latter group are with Ian. Meanwhile, Alvin becomes popular with the jocks and joins the football team, not realizing that the next game is during the concert. At the concert, Theodore and Simon tell the fans that Alvin didn't show up and that they can't perform, leading to a victory for the Chipettes. When Alvin finally shows up after the concert is over, he finds the auditorium empty and is ignored by his brothers at home.
Soon the Chipettes are hired, but learn that the concert they are to perform as an opening act for a Britney Spears concert at the Staples Center is on the same night as the school contest. Ian convinces them to blow off the battle and perform at the concert, but refuses to give the same credit to Jeanette and Eleanor that he gives Brittany. She demands that they all go together or not at all, until Ian threatens to send them to a barbecue restaurant (in a similar manner to the Chipmunks) unless they perform.
Before the Battle Of The Bands, Alvin receives a call from the Chipettes who tell him that Ian has locked them in a cage, so Alvin races off to rescue them while Simon tells Jeanette how to open the cage over the cell phone. Simon and Theodore are on the verge of going out to perform until the others arrive just in time to perform at the contest. The Chipmunks and the Chipettes perform together and receive the money for the music program. Dave, who had left the hospital upon learning that Toby was looking after the Chipmunks, returns during the contest happy to see his boys again. Meanwhile, Ian gets into more trouble at the concert of Staples Center he set up for the girls when he attempts to imitate them. After the contest, Dave allows the Chipettes to stay with them.
In two post-credits scenes, Dr. Rubin has the jocks scrape gum off from under the bleachers in the gymnasium, while Ian is thrown into a dumpster.
- Justin Long as Alvin Seville (Ross Bagdasarian, Jr. as Alvin's singing voice).
- Matthew Gray Gubler as Simon Seville (Steve Vining as Simon's singing voice).
- Jesse McCartney as Theodore Seville.
- Christina Applegate as Brittany (Janice Karman as Brittany's singing voice).
- Anna Faris as Jeanette (Janice Karman as Jeanette's singing voice).
- Amy Poehler as Eleanor (Janice Karman as Eleanor's singing voice).
- Zachary Levi as Toby Seville
- David Cross as Ian Hawke
- Jason Lee as David "Dave" Seville
- Wendie Malick as Dr. Rubin
- Anjelah Johnson as Julie Ortega
- Kathryn Joosten as Aunt Jackie
- Kevin G. Schmidt as Ryan Edwards
- Chris Warren, Jr. as Xander
- Bridgit Mendler as Becca Kingston