During Dor's annual "Royal Soup Day," sailor Pietro arrives alongside his curious rat companion, Roscuro, in a charming medieval French kingdom famed for its delicious soups. The kingdom's esteemed culinary expert, Chef Andre, skillfully creates his famed dishes with the aid of Boldo, a magical culinary genie who resides in his pot. However, tragedy strikes when Roscuro accidentally falls into Queen Rosemary’s soup during a royal banquet, causing her untimely death due to a heart attack. Overwhelmed by remorse, Roscuro soon finds himself abandoned in the castle's dungeons as Pietro departs.
In his dismal situation, Roscuro seeks refuge with the enigmatic Botticelli Remorso, the leader of Dor’s rat populace. Struggling to fit into their combative culture, he watches as grief-stricken King Philip issues a decree that bans all soup-related activities and declares rats illegal. This prohibition devastates Andre, cutting him off from his culinary passion and causing Boldo to abandon him. As the kingdom sinks into despair, Princess Pea, affected by her father's sorrow, quietly mourns their plight and her own increasing isolation.
Meanwhile, in a hidden mouse village within a neglected kitchen, Despereaux is born into the Tilling family. This newborn mouse stands out due to his large ears and unexpected behavior of not crying at birth. As Despereaux matures, his courage and curiosity starkly contrast the fearful temperament of the other mice. To adapt him to the expected "mouse" behavior, his brother Furlough takes him to the royal library, but instead of chewing the books, Despereaux finds himself captivated by their tales. He encounters Princess Pea and promises to unveil the conclusion of a story about a trapped princess.
However, when his parents, Lester and Antoinette, discover he has violated mouse law by conversing with a human, they turn him over to the mouse council to shift the blame away from themselves. The council makes a harsh decision to exile Despereaux for his bravery, sending him into the dungeons, presumed dead by the other mice. In the dark confines of the dungeons, he finds himself telling stories to the jailor, Gregory, who eventually leaves him alone. Captured by rats, Despereaux is thrown into an arena with a tabby cat, but Roscuro intervenes and requests him, presenting himself as a rescuer and feigning compliance with the rat's way of life.
As Roscuro struggles with his identity, he finds solace in tales of the outside world shared by Despereaux. In a bid to make amends for the chaos his actions caused, Roscuro ventures into Pea's chamber to apologize but is met with hostility, prompting him to harbor feelings of vengeance. He enlists the help of Miggery "Mig" Sow, Pea’s young maid who aspires to be a princess, convincing her that kidnapping Pea could help her achieve her dream. After Mig drags Pea to the dungeons, Roscuro betrays her, imprisoning her.
Understanding the peril Pea faces, Despereaux attempts to alert the King, who remains too consumed by sorrow to listen. Turning to his mouse family for help, he is shunned and mistaken for a ghost due to his floury appearance. Desperate, he rings the town bell, reminiscent of happier soup-making days for Andre, compelling him to revive his culinary arts and subsequently, rekindling Boldo’s loyalty. With agonizing hope, Despereaux seeks assistance from Andre and Boldo, but unexpected chaos ensues as Andre becomes distracted by a brewing thunderstorm triggered by the aroma of soup wafting skyward. Boldo heroically sacrifices himself, enabling Despereaux to reach the arena.
Inside the arena, witnessing the sorrow in Pea's azure eyes, Roscuro begins to regret his misguided quest for revenge; yet Botticelli, in a fit of anger, signals the rats to consume Pea. Despereaux bravely releases the cat to fend off some of the attacking rats while simultaneously defending Pea from imminent danger. As Botticelli corners Despereaux, a ray of sunlight shines through, stunning the light-sensitive sewer rats. Roscuro uses a spotlight to reflect the sunlight onto Botticelli, forcing him into the cat's clutches, while Pea ingeniously traps both of them, leaving Botticelli to face his fate as the other rats flee.
In the joyful aftermath, Mig is reunited with Gregory, revealed to be her estranged father, and they return to a simpler life on the farm. Meanwhile, King Philip emerges from his grief, reinstating soup and rats in the kingdom. As joy returns and the sun shines over Dor once again, Roscuro sets sail back to his former life and Despereaux embarks on a grand adventure, inspiring the mice to embrace bravery just as he has.
