Increased intestinal permeability is thought to underlie the pathogenesis of food allergy. We explore the mechanism responsible for changes in the morphology and function of the intestinal barrier using a rat model of food allergy, focusing on the contribution of intestinal microbiota. Juvenile–young adult rats were sensitized with ovalbumin and treated with antibiotics or probiotics (Clostridium butyricum and Lactobacillus reuteri), respectively. The serum ovalbumin-IgE levels, intestinal permeability, histopathological features, tight junction (TJ)-associated proteins, Th2 cytokines, and gut microbiota in feces were analyzed in each group. Sensitized rats showed an increase in ovalbumin-IgE levels and intestinal permeability with gut mucosal inflammation, whereas rats that received probiotics were only mildly affected. Rats given ovalbumin, but not those given probiotics, showed a reduction in both TJ-related protein expression and localization. Th2 cytokine levels were increased in the sensitized rats, but not in those given probiotics. TJs in rats treated with ovalbumin and antibiotics were disrupted, but those in rats administered probiotics were undamaged. Clostridiaceae were increased in the probiotics groups, especially Alkaliphilus, relative to the ovalbumin-sensitized group. Gut microbiota appears to play a role in regulating epithelial barrier function, and probiotics may help to prevent food sensitization through the up-regulation of TJ proteins.