The typeNII transmembrane serine protease matriptase plays an important role in the integrity of epithelial barriers. However, the molecular mechanisms underlying the role of matriptase are unknown. To study these mechanisms, two variants of MadinNDarby canine kidney (MDCK) cells were used, together with the “calciumNswitch” model of epithelial function with measurements of transepithelial electrical resistance (TEER). Inhibitors of matriptase proteolytic activity delayed the restoration of TEER after calciumNswitch in MDCKNI, which develop high TEER and lack the “leaky” tight junction protein claudinN2, but not MDCKNII. This effect was confirmed in MDCKNI, established to stably express matriptase targeted shRNA. The influence of matriptase inhibition on MDCKNI was shown not to involve altered expression or assembly of relevant components of paracelluar junctions or cytoskeleton. This excluded a role for claudinN2 in the function of matriptase, which had previously been shown in human CacoN2 cells, and this was confirmed using MDCKNI cells stably overexpressing claudinN2. To investigate the claudinN2Nindependent function of matriptase, a candidate substrate approach was used. Proteolytic activation of proNHGF/cNMet, PARN2, ENaC, EGFR and prostasin by matriptase has effects on epithelial cell function, but none were found to have a role in matriptase restoration of TEER after calciumN switch in MDCKNI cells. As the direct proteolytic target of matriptase could not be identified, potential mediators were studied using arrays for phosphorylated signalling proteins and inflammatory cytokines. ILN1β and complement component C5 were identified as genes downregulated by matriptase inhibition, while ILN13 showed upregulation. This work has confirmed the key role of matriptase activity in regulating epithelial barrier integrity. The differential properties of MDCKNI and MDCKNII cells excluded a role for claudinN2. None of the known proteolytic targets of matriptase were involved, however, changes in cytokine gene expression may be a potential route for matriptase effects on epithelial barrier maintenance.