Apical exposure of the intestinal epithelium to serine proteases results in an increase in transepithelial electrical resistance (TER); however the underlying mechanism(s) governing this response are unknown. We aimed to determine the requirement for proteolytic activity, epidermal growth factor receptor (EGFR) activation, and downstream intracellular signalling in initiating and maintaining enhanced barrier function following protease treatment. Apical stimulation with trypsin and matriptase significantly increases TER of polarized intestinal epithelial monolayers. Proteolytic activity by proteases is required to initiate and maintain protease-mediated increased TER. Matrix metalloproteinase (MMP)-independent EGFR activation is essential to the sustained phase of the protease response; Src kinases may mediate EGFR activation. Phosphoinositide-3 kinase (PI3K) and ERK1/2 signalling are important in reaching a maximal increase in TER following protease stimulation; however, their upstream activators are yet to be determined. Protein kinase C (PKC)ζ activity is important for epithelial barrier maintenance but likely not involved in protease-mediated increased TER. Our data show a requirement for ongoing proteolytic activity, EGFR transactivation, as well as downstream PI3-K and ERK1/2 signalling in protease-mediated barrier enhancement of intestinal epithelial cells. The enhanced barrier function mediated by proteases may contribute to novel therapeutic targets for intestinal disorders characterized by disrupted epithelial barrier function.