| Spoiler warning: The following contains plot details about|
the entire movie.
The Mayor finds out from Dr. LaRue (Isla Fisher) that Whoville will be destroyed if Horton does not find a "safer, more stable home." Horton resolves to place the speck atop Mt. Nool, the safest place in the jungle. The head of the jungle, the Sour Kangaroo (Carol Burnett), disbelieving Horton's beliefs, and resenting him for overshadowing her authority attempts several times to demand that Horton give up the speck, but Horton ignores her demands. Also taking force toward Horton are the Wickersham Brothers, a group of monkeys who love making misery. Eventually, the Sour Kangaroo, upon learning Horton's ignorance, enlists a vulture named Vlad Vladikoff (Will Arnett) to get rid of the speck by force.
Vlad manages to steal the clover away from Horton and drops it into a massive field of identical pink clovers, causing an apocalyptic tremor in Whoville. After unsuccessfully picking nearly 3,000,000 clovers, Horton eventually recovers the clover (exactly the 3,000,000th clover). The Sour Kangaroo eventually finds out that Horton still has the speck, and decides to rally the jungle community into fighting Horton, saying that Horton's goal will lead to anarchy. Upon cornering him, the Sour Kangaroo offers Horton an escape from punishment by renouncing Whoville's existence. When Horton refuses, in spite of his heartfelt speech, she orders the animals to rope and cage him, and to have the speck and the Whos destroyed in a pot of boiling beezlenut oil. The Mayor enlists all of his people to make noise by shouting "We are here!", as well as playing a variety of instruments, so the animals may hear them, assisted by Jojo's "Symphonophone", an invention which creates a huge musical contribution, but still fails to penetrate the surface of the speck. As Horton lies beaten and captured, the Sour Kangaroo easily takes the clover, and releases it. Meanwhile, JoJo grabs the horn used to project Horton's voice, runs up the highest tower and yells "YOPP!", breaking through the sound barrier just seconds before the speck hits the oil.
The Sour Kangaroo's son, Rudy (Josh Flitter) grabs the clover and returns it to Horton. The animals finally hear the Who's and realize the truth and isolate the Sour Kangaroo for tricking them. While being praised for his integrity by his neighbors, Horton even forgives the devastated Sour Kangaroo, and she befriends him with a makeshift umbrella for Whoville. Here, the people of Whoville and the animals of Nool gather in song and recite the chorus from "Can't Fight This Feeling". The film ends with the narrator revealing that the Jungle of Nool, or rather Earth, is just one speck, like Whoville, among numerous others, floating in outer space.
- Jim Carrey as Horton, an outgoing, big-hearted, loving, sweet, and thoughtful elephant. Horton has no tusks, lives by himself and possesses acute hearing abilities.
- Steve Carell as Mayor Ned McDodd, the mayor of Whoville. He has 96 daughters, 1 son named JoJo and his wife Sally.
- Carol Burnett as The Sour Kangaroo, The film's main antagonist, she thinks that Horton is a fool for thinking that there is life on a speck of dust.
- Will Arnett as Vlad Vladikoff, a vulture hired by the Sour Kangaroo to steal Horton's clover.
- Seth Rogen as Morton the mouse, Horton's best friend in the Jungle of Nool
- Amy Poehler as Sally O'Malley, Ned's wife and mother to Jojo and 96 daughters
- Dan Fogler as the Councilman and Yummo Wickersham
- Jesse McCartney as JoJo, Ned's son
- Isla Fisher as Doctor Mary Lou LaRue, a teacher at Who U.
- Laura Ortiz as Jessica
- Jonah Hill as Tommy
- Jaime Pressly as Mrs. Quilligan, Jessica's mother
- Niecy Nash as Miss Yelp
- Selena Gomez as Helga, one of the mayor's daughters
- Josh Flitter as Rudy Kangaroo
- Joey King as Katie, a cute little baby yak
- Bill Farmer as Willie Bear, additional voices
- Charles Osgood as The Narrator
- Bloom Loom as Who Woman/Joan