The film opens with a whimsical portrayal of Dr. John Dolittle (Robert Downey Jr.) set in 19th century Britain, narrated by Poly the parrot (Emma Thompson). Renowned for his unique gift of communicating with animals, Dolittle once traveled the globe with his beloved wife Lily ([Kasia Smutniak]) to rescue and care for injured creatures. Tragedy strikes when a sailing expedition with Poly ends in disaster, leading to Lily's untimely demise in a fierce storm. A heartbroken Dolittle isolates himself from the outside world, shutting down his sanctuary and mourning his loss.
As years roll by, a young boy named Tommy Stubbins (Harry Collett) embarks on a hunting trip with his uncle and cousin. Nervous and inexperienced, he accidentally injures a squirrel named Kevin (voiced by Craig Robinson), and instead of finishing him off as commanded, he takes the creature to seek help. Poly, noticing the boy's plight, guides him to Dolittle's sanctuary, where he encounters Yoshi the polar bear (John Cena), causing a humorous mishap that leads him to meet Lady Rose (Carmel Laniado), who has urgent news from Queen Victoria (Jessie Buckley).
Inside Dolittle's home, amidst a game of chess with his animal companions, including Chee-Chee (voiced by Rami Malek), the timid gorilla, Dab-Dab (Octavia Spencer), the scatterbrained duck, and Jip (Tom Holland), the bespectacled dog, the unsettling news reaches Dolittle. Princess Rose informs him that Queen Victoria is gravely ill, poisoned by deadly nightshade, and the only antidote—a rare Eden Fruit—grows on a faraway island. Despite his reluctance to rejoin civilization, Dolittle eventually agrees to venture out on this quest, spurred on by Poly's encouragement.
Setting off aboard a makeshift ship alongside Plimpton ([Kumail Nanjiani]), a nervous ostrich, Poly, Yoshi, Kevin, Jip, and Dab-Dab, Dolittle arrives at Buckingham Palace. There, he encounters his former rival, Dr. Blair Müdfly (Michael Sheen), and realizes the urgency of his mission to save the Queen. In a moment of self-doubt, Dolittle sends the eager Stubbins home, not wanting to involve him in the dangerous adventure.
The next day, determined to assist Dolittle, Betsy the giraffe ([Selena Gomez]) and Tutu the fox ([Marion Cotillard]) sneak in to help Stubbins escape his home, just as Dolittle's ship sails away. With quick thinking, they catch up, leading to a thrilling boarding of the ship, and Stubbins becomes absorbed in the world of animal communication, earning Dolittle’s recognition.
Meanwhile, Dr. Müdfly and his crew pursue them, prompting Dolittle to seek the aid of a whale to quicken their journey. However, obstacles arise when their map fails them, leading to a stop at an island controlled by King Rassouli ([Antonio Banderas]), Lily’s father, who possesses crucial information about the tree's location. Disguised and stealthy, Dolittle and Stubbins venture into Rassouli's lair only to be captured.
In a dramatic showdown, Dolittle faces off against Barry ([Ralph Fiennes]), a menacing tiger with unresolved issues, while Stubbins is left in the hands of bandits. Through teamwork, with the help of the other animals, Dolittle escapes, but the villainous Müdfly manages to steal the key to their mission—the journal leading to the Eden Fruit—and destroys the ship.
Despite seemingly insurmountable odds, a moment of camaraderie occurs between Yoshi and Plimpton, revealing their shared experiences of neglect. Rassouli, overhearing Dolittle’s feelings of failure, gifts him an old ship, urging him to honor Lily’s memory by continuing his quest. With renewed determination, Dolittle sets off with the help of the ocean’s whales to track down Müdfly.
As they arrive at the island where the fruit is located, tensions mount as Müdfly’s men have beaten them there. A fierce battle erupts with the appearance of a fearsome dragon. Dolittle, acting with compassion, saves Müdfly and learns an invaluable lesson about kindness, all while facing perils of his own. He cleverly uses his knowledge to alleviate the dragon's pain, earning the precious fruit needed to save the Queen.
Rushing back to the palace with minutes to spare, Dolittle and the animals face the nefarious Lord Badgley, who is revealed to be the architect of the poisoning plot. With Stubbins successfully administering the fruit to the Queen, order is restored, and Badgley faces justice.
In a joyful conclusion, Dolittle embraces his new role as a mentor to Stubbins, reopening the sanctuary to the public in a tribute to Lily's legacy. In a humorous post-credits scene, we see Müdfly still trapped, humorously struggling to connect with bats in his new surroundings.
