Despereaux is a tiny, sickly mouse born with unusually large ears and a fascination for light and music. Rather than learning to scurry and scavenge, he spends his time reading fairy tales in the castle library and listening to the king play music. His non-conformist behavior quickly draws the critical attention of his family and the broader mouse community.

Princess Pea is the twelve-year-old heir to the Kingdom of Dor. She carries a profound sadness following her mother's sudden death, a tragedy that prompted her father to outlaw soup. Despite the mourning period hanging over the castle, she retains a sharp sense of empathy and kindness toward those she encounters.

Chiaroscuro, who prefers the nickname Roscuro, is a rat born in the castle's dark dungeon. Unlike his peers, he harbors an intense obsession with bright light and beautiful things. He constantly struggles between his natural rat instincts for darkness and his unusual desire to experience the illuminated upstairs world.

Miggery Sow is a peasant girl named after a prize pig. Following her mother's death, she is sold into servitude and subjected to years of physical abuse, leaving her with severe hearing loss. Despite her grim circumstances and lack of natural aptitude for castle chores, she holds tightly to an impossible dream of becoming a princess.

Botticelli Remorso is a seasoned dungeon rat who thoroughly enjoys his identity and the dark environment he inhabits. Missing one ear and wearing a necklace made of a heart-shaped locket and mouse whiskers, he believes the meaning of life is causing suffering. He actively attempts to mentor younger rats in the ways of cruelty and manipulation.

Gregory is an elderly man tasked with guarding the castle's dungeon. He navigates the pitch-black maze by keeping a rope tied around his ankle to prevent himself from getting lost. He possesses a deep appreciation for stories and memories of the past, often requesting tales from prisoners in exchange for small mercies.

Hovis is an older mouse tasked with preparing condemned mice for the dungeon. Though his job makes him an administrator of death within the community, he performs his duties with quiet regret. He possesses an uncharacteristic appreciation for fairy tales and courtly romance, which allows him to sympathize with rule-breakers.

Antoinette is a French mouse who places a high value on her physical appearance and dramatic flair. She frequently wears heavy makeup and considers the hardships of motherhood to be a personal burden. She is quick to complain about her youngest son's unusual traits rather than offering him maternal support.

Lester is a respected member of the mouse community who cares deeply about adherence to rules and social norms. When his son begins displaying strange behaviors, Lester prioritizes the community's laws over his family bonds. He serves the Mouse Council by beating the thimble-drum to summon the elders for trials.

Furlough is Despereaux's older brother and a model citizen of the castle's mouse community. He is highly concerned with typical mouse activities like scavenging for crumbs and hiding from humans. When he witnesses his brother breaking fundamental rules, he chooses loyalty to mouse law over family loyalty.

Merlot is Despereaux's older sister. Like the rest of her family, she is highly conventional and struggles to understand her sibling's unusual behavior. She tries to teach him the proper way for a mouse to interact with books, viewing them as a food source rather than a medium for stories.

King Phillip is the ruler of the Kingdom of Dor and a man completely consumed by grief. Following the sudden death of his wife, he makes emotional and highly impractical decrees, such as outlawing all soup and kettles across the land. His heartbreak makes him fiercely protective of his daughter and entirely dismissive of rodents.

Cook is the head of the castle's kitchen staff. She is a pragmatic, hard-working woman who frequently resorts to physical discipline when managing incompetent servants. Despite the king's strict laws against her life's passion, she secretly continues to practice her culinary arts.

Queen Rosemary is the late monarch of the Kingdom of Dor. Her defining characteristic was her profound love for soup, which she considered the ultimate comfort food. Her sudden death by heart attack during a royal banquet sets off a chain of strict laws and deeply affects the emotional state of the entire kingdom.

"Uncle" is the harsh, unnamed farmer who buys a young orphaned girl to use as unpaid labor. He is an abusive and dismissive man who regularly hits the girl around the ears when she makes mistakes. He actively crushes her dreams, insisting that no one cares what she wants.

The Most Very Honoured Head Mouse is the strict leader of the castle's rodent population. He upholds the rigid laws of his community without exception, demanding total conformity from all mice. He views any interaction with humans as a dangerous betrayal that warrants severe punishment.