In Victorian era Britain, Dr. John Dolittle is a Welsh veterinarian with the ability to communicate with animals. After his wife, Lily, dies at sea, Dolittle retreats from human society and only tends to animals. A boy, Tommy Stubbins, accidentally wounds Kevin the squirrel and is guided to Dolittle’s help by Polynesia, a macaw.
Queen Victoria summons Dolittle to cure her of a deadly sickness. He refuses until Poly persuades him that he must begin to reconnect with fellow humans. He finds the Queen has been poisoned by kind of nightshade added to her tea. The only cure is the magical fruit of Eden from the faraway land of Lily’s birth.
Poly, Betsy the giraffe, and Tutu the fox help Tommy escape from his home, and he joins Dolittle’s crew including Kevin, monkeys Elliot and Elsie, Chee-Chee the gorilla, Yoshi the polar bear, Plimpton the ostrich, Dab-Dab the duck, and Mini the sugar glider. They put to sail for the cure while evading Dolittle’s lifelong rival, Dr. Blair Müdfly. Dolittle leaves his lurcher Jip and a walking stick named Sticks behind to guard the Queen while he’s traveling. Dolittle’s boat is attacked by Müdfly and they escape by harnessing a whale who pulls the boat to safety.
They continue to Lily’s island where, attempting to steal Lily’s directions to the fruit tree, Dolittle is apprehended by Lily’s father King Rassouli and is locked in a cage with Barry, a moody tiger looking for his mother’s approval. Just as Dolittle is to be killed, a newly-courageous Chee-Chee incapacitates Barry. Dolittle and Tommy escape but lose Lily’s journal to Müdfly and have their ship destroyed. Rassouli loans Dolittle a boat to honour his daughter.
Trailing Müdfly, Dolittle and company meet Ginko-Who-Soars, a dragon who guards the cure tree. Ginko attacks, causing Müdfly to fall down a hole, but soon collapses from internal pain. Dolittle diagnoses and cures the dragon who gratefully shows Dolittle the tree.
Dolittle returns in time to heal the queen. Sticks reveals that Lord Thomas Badgley, one of the Queen’s chairmen, poisoned her in order to take the crown for himself, and it was he who ordered Müdfly to foil Dolittle’s quest. The Queen has him arrested for treason, and thanks Dolittle for saving her.
Dolittle takes on Tommy as his apprentice and re-opens his sanctuary.