India Opal Buloni is a young girl who lives with her father, a preacher, in a trailer park. On a grocery run, Opal finds the store in disarray as a dirty dog runs up and down the aisles. Instead of letting the dog go to the pound, Opal claims the dog as her own, and names him after the grocery store: Winn-Dixie. Opal takes him home to her father in hopes that she can keep him; Winn-Dixie wins over the preacher with his wide smile and charming personality.
Opal and the preacher quickly realize that this dog does not like to be left alone: he pulls off the couch cushions, unrolls the toilet paper, and howls if he is tied outside. Opal and the her father have no choice but to bring Winn-Dixie to church with them. This results in a bit of chaos at first, but once again Winn-Dixie wins the heart of everyone as he catches a mouse, but gently holds it in his teeth, careful not to hurt it.
As he begins to look healthy, Opal decides to get him a new leash and collar from Gertrude’s Pets. She cannot afford the items and asks Otis, the pet store owner, if she can work for him to pay for her desired items. Not surprisingly, Winn-Dixie wins over Otis (and his pet parrot Gertrude) and Opal is given a job. Opal is thrilled with the new friends she has made thanks to Winn-Dixie.
One day Opal rides her bike past Dunlap and Stevie Dewberry’s house; they begin following her and whispering behind her back. The brothers tease her about getting close to a house that is supposedly inhabited by a witch. Before she can stop him, he runs straight into the witch’s yard. Opal decides she loves her dog more than she fears witches, so she goes in after him. Here, Opal meets the “witch”, Gloria Dump, a crinkly, kind, old woman with bad vision. Gloria asks Opal to talk about herself so that she can see her with her heart. Opal is thrilled to tell her everything: how she misses her mom, how her dad hides in his "turtle shell", how she got a job, how she was invited to a party, and how the Dunlap boys are mean to her. Gloria listens thoughtfully to everything Opal shares.
That night there is a terrible thunderstorm that wakes Winn-Dixie. Opal and the preacher determine that he has a pathological fear of thunderstorms; they realize that they must watch Winn-Dixie carefully during thunderstorms so that he doesn’t run away.
One day at the library, Miss Franny Block tells Opal, Winn-Dixie, and Amanda about her great-grandfather and the Civil War. Miss Franny gives them all a Littmus Lozenge, a candy her great-grandfather invented. Everyone tries one and agrees that the candy is a mixture of root beer, strawberry, and sorrow. Opal recognizes that everyone feels some sadness, from the past, inside them. She also realizes that she cannot judge people for the mistakes they have made.
As Opal builds relationships with members of the community, she decides to have a party and invite all of her new friends. Just as everyone arrives to the party, a sudden, terrible downpour occurs. All the guests rush inside, trying to save the punch and the egg salad sandwiches. Opal had forgotten about Winn-Dixie and, with a feeling of dread, she runs outside to find him; she fears that he has run away. Opal and her father search and search, but soon her father says that it’s time to give up.
At this point, Opal is honest with her father for the first time. She blames him for living in his "turtle shell" and accuses him of letting her mother walk out on them. As a result, the preacher breaks down into tears over the loss of Opal’s mother, but Opal comforts him and recognizes that she and her father need each other. The two return to Gloria’s empty-handed. Winn-Dixie, however, had been hiding under the bed all along! Opal is thrilled and relieved that her dog is safe. The party continues on with Opal and her friends happily singing together.