DiCamillo explores several themes through her characters and their interactions. Opal, along with the other characters, grapples with issues like love, loss, and friendship. As these characters come together, they gain wisdom and learn valuable lessons about holding on, letting go, listening, and sharing. In the end, Opal realizes what truly matters.

Opal is an honest, humorous, and lonely young girl who fears disappointing her father. With Winn-Dixie by her side, she decides to ask him about her mother who left them. She then memorizes what her father tells her because, as she says, "I wanted to know those ten things inside and out. That way, if my mama ever came back, I could recognize her, and I would be able to grab her and hold on to her tight and not let her get away from me again."

Opal's desire is not just to remember her mother, but to soothe her own fears and feel safe and loved. Her father, absorbed in his work and his own pain, fails to recognize the reassurance his daughter desperately needs. Observing him with Winn-Dixie during one of the dog's episodes, Opal tells the reader, "I loved the preacher so much.... I loved him because he was going to forgive Winn-Dixie for being afraid. But most of all, I loved him for putting his arm around Winn-Dixie like that, like he was already trying to keep him safe."

Throughout the story, Opal expresses her loneliness and her longing for someone to hold on to. She talks to God about her loneliness; she adopts Winn-Dixie because he does not "belong to anybody;" and she takes him everywhere because being "left behind probably made his heart feel empty." Opal relates to this feeling, as she feels the same way. Her need to hold on to her mother's memory is strong, and she searches for ways to do it: memorizing things about her, collecting stories she hopes to one day share with her, and trying to be good so that no one else will leave her.

Opal's new friends also cling to various things. Miss Franny holds on to her library and the Littmus Lozenge candy to remind her of the past because all her friends and family are gone. Otis keeps his music to himself, fearing a return to jail. Gloria Dump hangs her empty whiskey bottles on a tree to remember past mistakes. Amanda, grieving her little brother, isolates herself from others. The preacher, too, suppresses his emotions, afraid of losing again if he loves too deeply.

Winn-Dixie guides the characters in releasing their pain. DiCamillo uses the dog to show them what they should cherish. However, like the Littmus Lozenge, which combines sweetness and sorrow, the characters must first acknowledge that life includes both happiness and suffering, and it involves both holding on and letting go. Miss Franny learns that she doesn't have to be lonely in her library surrounded by books. She discovers that sharing her stories can bring companionship. By sharing his music, Otis gains self-confidence and friends. Amanda begins to heal from her brother's death by allowing others to be her friends. Gloria, by imparting the wisdom she has gained, also makes friends and helps others realize what truly matters. Opal learns about letting go when she loses Winn-Dixie. "I was supposed to hold on to him," she says. But Gloria Dump responds, "There ain't no way you can hold on to something that wants to go, you understand?"

While frantically searching for Winn-Dixie, Opal and her father confront reality when the preacher says, "it's time to give up." In her desperation, Opal accuses him of giving up on her mother and letting her leave. Fortunately, Opal sees her father's pain and fear in his response, and she also recognizes his profound love for her, which was all she truly needed to know. Despite being a catalyst, Winn-Dixie also learns a new way to deal with his fears and, in doing so, teaches Opal another crucial lesson: letting go doesn't mean losing.