Ten-year-old India Opal Buloni (ANNASOPHIA ROBB) is in dire need of a friend. She's just moved to the small town of Naomi, Florida with her father (JEFF DANIELS), a Baptist preacher who holds his services in the local convenience store for lack of any proper space. While there are some kids around, they're too young such as Sweetie Pie Thomas (ELLE FANNING), seemingly stuck up like Amanda Wilkinson (COURTNEY JINES) or seem to revel in giving her a hard time like the young Dewberry brothers, Dunlap (NICK PRICE) and Stevie (LUKE BENWARD). With no mother -- she left when the girl was three -- and her dad being unable or unwilling to talk about her, Opal's future seems isolated at best.
Things change, however, when her dad sends her to the local grocery store for a few items. It's there that she witnesses the manager and others trying to corral a dog that's on the loose inside the store. When she hears the manager call for the pound, Opal lies that the dog is hers and - spotting the store's name at the last second -- calls him Winn-Dixie.
Although the manager of the trailer park where they live -- Mr. Alfred (B.J. HOPPER) -- says dogs aren't allowed there, Opal's dad reluctantly agrees to let her keep the dog temporarily until they find its owner. Despite that directive, she and Winn-Dixie soon become fast friends, with Opal discussing all of her feelings to the dog that ends up introducing the girl to some of the town's inhabitants.
They include Otis (DAVE MATTHEWS), a guitar playing drifter who's running the pet store and gives Opal a job in exchange for a dog collar; bookworm Franny Block (EVA MARIE SAINT) who runs the local library and has no family; and Gloria Dump (CICELY TYSON) who the Dewberry boys claim is a witch, but is really a nearly blind recluse and recovering alcoholic.
As she and Winn-Dixie meet and befriend all of them, Opal hopes to learn more about her absent mom, all while helping her dad and the others face and overcome their individual issues and problems.