The prevalence of food allergy has increased worldwide but the pathogenesis remains undefined and effective treatments has not been established. Transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive nonselective cation channel, is mainly expressed in epithelium of various organs. The present study investigated the role of TRPV4 in the pathogenesis of ovalbumin (OVA)-induced food allergy in mice. TRPV4 was mostly expressed in colonic epithelium. Repeated oral OVA challenge after sensitization induced food allergy, characterized by systemic allergic symptoms, diarrhea, upregulation of Th2-cytokines such as IL-4, IL-5, IL-13, and increase in serum OVA-specific antibodies, but all these responses were significantly augmented in TRPV4-deficient (TRPV4KO) mice compared with wild-type (WT) mice. Infiltration of CD11c-, CD117-, CD4-, and CD170-positive cells in the colon with OVA-induced food allergy was also enhanced in TRPV4KO mice compared with WT mice. Intestinal permeability determined by the FITC-dextran method was significantly increased in TRPV4KO mice compared with WT mice in normal and OVA-induced food allergy. Furthermore, the expression of adherence junction protein E-cadherin, tight junction protein claudin-3 and occludin in the colon was significantly lower in TRPV4KO mice than WT mice in normal and food allergy. These results suggest that epithelial TRPV4 protects OVA-induced food allergy. This response may be accounted for by suppressing the penetration of allergens via maintaining epithelial barrier functions.