AbstractEpithelial barrier dysfunction is involved in a large number of diseases, but the pathogenesis is unclear. Integrin alphavbeta6 (avb6) in involved in the maintenance of the mucosal homeostasis. We have investigated the role of avb6 in maintaining the epithelial barrier function. Using T84 monolayers cultures, transepithelial electric resistance (TER) and permeability to ovalbumin (OVA) were measured as indicators of functioning. The antigenicity of OVA collected from the Transwell basal chambers was assessed using OVA‐specific T cell proliferation. Knockdown of the avb6 genes increased the permeability of T84 monolayers to OVA, but did not affect TER. The deficiency of avb6‐related hyperpermeability in T84 monolayers could be compensated by adding exogenous avb6 to the culture. The OVA samples collected from the basal chambers had strong antigenicity as it markedly induced the antigen specific T cell proliferation. Addition of recombinant avb6 blocked increases in permeability of T84 monolayers to OVA induced by tumor necrosis factor‐α.