Epithelial permeability to ions and larger molecules in the gut is essential for fluid balance and its dysregulation contributes to intestinal pathology. We investigated the effect of digestive serine proteases on epithelial paracellular permeability. Trypsin, chymotrypsin and elastase elicited sustained increases in transepithelial resistance (R_TE) in polarised monolayers of three intestinal epithelial cell lines. This effect was reflected by decreases in paracellular conductances of Na⁺ and Cl^- and a concomitant decrease in permeability to 3000 MW dextran. The enzyme activities of the proteases were required, yet activators of known protease-activated receptors (PARs) did not reproduce the effect of these proteases on R_TE. A PKC isoform-specific inhibitor significantly reduced the trypsin-induced increase in R_TE while PKC activity was increased in cells treated with trypsin and chymotrypsin in comparison to control cells; this activity was reduced to control levels in the presence of the PKC-specific inhibitor. Ca⁺⁺ chelators and pharmacologic inhibitors of cell signalling support the role for PKC in the protease-induced effect. Finally, treatment with the serine proteases increased occludin immunostaining and zonula occludin-1 (ZO-1) co-immunoprecipitation with occludin in the detergent insoluble fraction of cell lysates, and these increases were ablated by pre-treatment with PKC specific inhibitor. This finding indicates increased insertion of occludin into the cell junctional complex. These data demonstrate a role for serine proteases in the facilitation of epithelial barrier function through a mechanism mediated by the PAR-independent activation of PKC.