After she experiences apocalyptic visions, Rei is attacked by a new species of Daimon that Sailor Moon is unable to fight after it breaks her Crystal locket, reversing her transformation. The Daimon is defeated by the returning Sailors Uranus and Neptune, who have not been seen since the adventure with Hercules, leaving Rei to despondently realize that a new conflict has begun.
During a benefit concert in Paris, France, David Seville (Jason Lee) is injured, leaving him recuperated there and asks his aunt (Jackie Seville) 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), Dave's first cousin once removed (Dave's Aunt Jackie is Toby's grandmother). 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, Simon is bullied by jocks, who Alvin makes friends with and visit the principal's office, only to discover that the principal (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 Spearsconcert 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 murder them in a similar manner to the Chipmunks.
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, the principal has the jocks scrape gum off from under the bleachers in the gymnasium, while Ian is thrown into a dumpster.