Wheat flour, as the most important source of food globally, is one of the most common causative agents of food allergy. This study aimed to investigate the effects of fermentation on wheat protein digestibility and allergenicity. Protein digestibility were evaluated using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and enzyme-linked immunosorbent assay. The effect of protein on intestinal permeability was investigated by Caco-2 cell monolayers. Co-culture fermentation with Pediococcus acidilactici XZ31 and yeast leads to improvement in digestibility of wheat protein compared to single strain fermentation. Fermentation leads to a decrease in albumin/globulin antigenicity and an increase in gluten R5 reactivity, with the most significant changes in the co-culture group. Digestion strengthen the decrease of protein antigenicity and counteracts the difference in antigenicity induced by fermentation between groups. However, pretreatment with P. acidilactici XZ31 reduces the amount of allergens across Caco-2 monolayer and attenuates the gluten-induced increase in permeability of Caco-2 cell monolayer by reducing actin polymerization and villous atrophy. Co-culture fermentation reduces gluten-induced cell monolayer damage to a greater extent than P. acidilactici XZ31 monoculture. These results gives valuable insight into the effects of P. acidilactici XZ31 fermentation on the allergenicity and toxicity of wheat proteins, which contribute to promoting the application of multi-strain leavening agent in hypoallergenic and gluten-free wheat products.