In fact, he finds out the speck harbors the city of Who-ville and all its inhabitants, led by Mayor Ned McDodd (Steve Carell). He has a loving wife, Sally (Amy Poehler), 96 daughters (Selena Gomez) who all have names that start with the letter H, and one son named JoJo (Jesse McCartney), who, by Who custom, is next in line for the mayoral position. JoJo does not want to become mayor, which leads him to become sullen and mute, despite Ned's giving him extra attention.
The Mayor finds out from Dr. Larue (Isla Fisher) that Who-ville will be destroyed if Horton does not find a "safer more stable home." So Horton resolves to place the speck atop Mt. Nool, the safest place in the jungle. This outlook earns Horton nothing but ridicule from the inhabitants of Nool, especially from the strict official of the jungle, the Sour Kangaroo (Carol Burnett), who tries to get Horton to give up the speck, so as not to put supposedly ridiculous ideas into the heads of the children. Ever faithful to his motto, "A person's a person, no matter how small," Horton refuses. Also taking force toward Horton are the Wickersham brothers (Frank Welker and Dan Castellaneta), a group of bullying monkeys who love making misery.
All the small incidents that Horton experiences on his trek across the jungle have a catastrophic effect on Who-ville. He almost falls off a rickety bridge over a gaping chasm with a river of dangerous crocodiles at the bottom, which causes a dentist's needle to accidentally slip into the Mayor's arm while getting a root canal. When Horton left the clover outside overnight, it frosted over, which created winter in the summer down in Who-ville. As the other Whos become suspicious, the Mayor finally reveals the truth, but at first, the Whos do not believe him any more due to the Chairman than the animals believe Horton.
In the meantime, the Kangaroo has enlisted a nefarious buzzard named Vlad Vladikoff (Will Arnett) to get rid of the speck by force. Vlad manages to steal the clover with the speck on it, flee from a chasing Horton and drop it from hundreds of feet into a valley full of nearly identical clovers, (the one holding the speck has a striped stem). The impact nearly demolishes Who-ville like an earthquake. Horton painstakingly picks 2,999,999 clovers through the field and finally finds it "on the 3 millionth flower." The earthquake, combined with hearing Horton's voice through the drain pipe, is enough to convince the rest of the Whos that the mayor is not crazy, and they all tell Horton they believe in him.
The Kangaroo finds out that Horton still has the speck, and, as her patience completely runs out, organizes a mob by telling lies to get rid of the speck once and for all. The animals plan to rope and cage Horton, but the Kangaroo turns this into a chance for attention, and offers Horton an ultimatum: give up the speck and "admit" he was wrong and that she was right, or pay the price. Despite a heartfelt speech from Horton that clearly touches the animals, Kangaroo still takes this refusal as an insult to her authority, orders them to proceed, and drop the speck into a pot of boiling beezlenut oil to "teach him not to make up stories of people on specks!"
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 can hear them. JoJo runs off to Who-ville's abandoned Star-Studying Tower and soon Ned takes off after him. Inside, he reveals his ingenious invention: the Symphony-Phone, a giant machine that serves as an orchestra, and proceeds to add it to the mix of sounds. Still, the sound is not loud enough. The animals do not hear anything and the Kangaroo, who has had Horton caged, takes the clover, holds it over the oil and releases it. In a last-ditch effort to make Whoville heard, JoJo grabs the horn used to project Horton's voice, runs up the highest tower and yells "YOP!" A sound wave emerges and ripples up to the already pressured clouds and collides with them, causing the clouds to break and the sound to come through.
Hearing the Whos' cries, Rudy (Josh Flitter), the Kangaroo's son (who has been in his mother's pouch throughout the film despite being old enough to be out and too large for her pouch), grabs the clover before it hits the oil and returns it to Horton, refusing his mother's orders to return to her pouch. The animals finally realize the atrocity they almost committed. The Kangaroo is miserable for her behavior, but Horton forgives her, and offers his friendship, which the Kangaroo accepts. At the end of the film, everyone helps Horton carry the speck up to the top of Mt. Nool. After a big number of the cast singing REO Speedwagon's "Can't Fight This Feeling", the camera zooms out, revealing that along with numerous other worlds in our universe, the jungle of Nool is just one speck among numerous others like our planet.