Dr. John Dolittle is a Welsh veterinarian who possesses animal communication skills and lives in Victorian-era Britain. Dolittle focuses on caring for the animals at his sanctuary, which Queen Victoria gave him. He married Lily, a fearless explorer. They traveled the globe on great adventures, defending creatures who could not defend themselves. One day, Lily went on an adventure and never returned. He withdraws from human civilization after his wife, Lily, perishes at sea.

Years later, while out with his hunting father, Tommy Stubbins, a young child, inadvertently injures Kevin, a red squirrel, by saving ducks. A macaw Polynesia leads him to Dolittle's residence in the hopes that his presence will facilitate Dolittle's reconciliation with humans. Dolittle comes to know about the human intruder inside his sanctuary and gets alerted by his family (i.e., animals)

Simultaneously, Queen Victoria sends her maid of honor, Lady Rose, to ask Dolittle for assistance after she becomes very sick.  Initially, he ignores to come along with her to meet Queen. Dolittle treats Kevin first. Later, he agrees to meet Victoria after Poly tells him he will forfeit his house to the Treasury if the queen passes away. Dolittle also asks Tommy to return to his home, but he is willing to go with him since he likes animals and Dolittle cares for them. 

Dolittle discovers Dr. Blair Müdfly, his lifelong nemesis, tending to Victoria at her palace. He examines her personally in front of Müdfly and House of Lords member,  Lord Thomas Badgley and finds that she was poisoned with nightshade, for which there is only a magical cure, a fruit from Eden Tree Island.

Dolittle sets out on an expedition to deal with this, leaving his stick insect Styx, and lurcher Jip to watch over the Queen in his absence. He refuses to bring Tommy on the trip, even if he brings Chee-Chee the gorilla, Yoshi the polar bear, and Poly with him.

Betsy the giraffe and Tutu the fox are sent to fetch Tommy by Poly, who will not allow him to be left behind. Poly makes Tommy take some risks and makes him be there on their journey. Also, Poly convinces Dolittle to take the boy along, as he needs help from one of his kind. After learning how to communicate with animals, Dolittle offers assistance to Tommy. Müdfly starts chasing after Dolittle to obstruct his progress. With the help of a whale, they speed across Müdfly.

As Eden Tree cannot be found on any map, they need Lily’s journal to locate the island. So Dolittle sends his expedition to Monteverde, which is governed by Lily's father, King Rassouli, to locate the fruit and retrieve her journal. He thinks it will help point the way to the location of the fruit. But when Dolittle tries to steal the journal, Lily’s father, Rassouli, catches him and puts him in with Barry, a cranky tiger.

Dolittle stalls Barry until Chee-Chee discovers him and musters the confidence to overcome it as Tommy tries to get the journal. Despite the group's acquisition of the logbook, Müdfly takes it and blows up their ship. When Rassouli finds out that Dolittle is missing Lily and is doing his voyage in her honor, he lends him a ship so he may go after Müdfly. On the other side, the Queen’s health is worsening day by day. To get the journal back, they track Müdfly’s ship with the help of whales.

When they finally arrive on the island of the Eden Tree, Müdfly also gets them there. The dragon Ginko-Who-Soars is there to guard it. Müdfly falls down a hole when it attacks the groups, but it eventually passes out from internal anguish. Dolittle finds that something is wrong with this dragon and explains what it has been going through. The dragon allows Dolittle to diagnose it, and he cures the dragon.

Ginko, the dragon, gives Dolittle access to the tree and its fruit as a thankful reward. After returning in time to heal Victoria with the help of Styx, Dolittle confesses that Badgley poisoned her to seize the throne for himself. Thanking Dolittle for saving her life, the queen has him transported to the Tower of London on charges of treason.

After returning home, Dolittle appoints Tommy as his apprentice and reopens it to the public. Finally, Dolittle and his family are honored by the Queen for their help.