A 10-year-old girl named India Opal Buloni has just moved to a trailer park in the small town of Naomi, Florida, with her father, who is known as The Preacher because he preaches at the local church. Her mother, Benjean-Megan, abandoned them when Opal was three. She describes the preacher as a turtle, always sticking his head into his shell, and never wanting to come out into the real world. This is most likely because of how sad he is about her mother, with whom he is still in love.
While in the supermarket, Opal sees a scruffy dog wrecking the store and decides to take him home, naming him Winn-Dixie after the supermarket chain. Miss Franny Block, a librarian, shares great stories about her past, including one about her great-grandfather, whose family members died while he was fighting for the South in the Civil War. He invented Litmus Lozenge candies, which tasted like root beer and strawberry but included a secret ingredient—melancholy. Anyone who tasted the candies tasted sweetness mixed with sadness. Opal learns that her sour-faced neighbor, Amanda Wilkinson, lost her younger brother Carson when he drowned in the town lake the previous summer. She vows to be nicer to her from then on.
Opal finds a dog collar that she wants to buy for Winn-Dixie, but she has no money and decides to work for the pet store to earn it. Otis, a worker at Gertrude's Pets, is unwilling to hire Opal as a cleaning girl, but she comes to work. When Opal and Winn-Dixie step into the store, the animals panic when they see the big dog. Otis plays his guitar to calm them. Opal learns that Otis once went to jail for battering a police officer who tried to confiscate his guitar after the officer told Otis he could not play music on the street because he was disturbing others.
While Opal is riding her bike and Winn-Dixie runs ahead, they meet a woman named Gloria Dump. She and Opal become good friends. Opal and Gloria, a recovering alcoholic, decide to host a small party, inspired by the one in Gone with the Wind, inviting everyone they know. In the process, Opal becomes a friend to her former enemies, the brothers Stevie and Dunlap Dewberry. She also invites Amanda Wilkinson and Sweetie Pie Thomas, a younger girl who has no pet, and so has fallen in love with Winn-Dixie. Otis and Miss Franny Block are also invited.
Opal and Gloria set up everything outdoors, but it starts to rain, so they bring the party indoors. Opal can't find Winn-Dixie anywhere, even after searching the town. Ten minutes later she returns to Gloria's home to discover that Winn-Dixie has been there all the time, hiding because he is scared of storms. The book ends with Otis playing his guitar and everyone singing one of The Preacher's songs.
