The enteric microbiota contributes to the pathogenesis of inflammatory bowel disease but the pathways involved and bacterial participants may vary in different hosts. We previously reported that some components of the human commensal microbiota, particularly Clostridium perfringens, have the proteolytic capacity for host matrix degradation and reduce transepithelial resistance. Here, we examined the C. perfringens-derived proteolytic activity against epithelial tight junction proteins using human intestinal epithelial cell lines. We showed that the protein levels of E-cadherin, occludin and JAM-1 decrease in colonic cells treated with C. perfringens culture supernatant. E-cadherin ectodomain shedding in C. perfringens-stimulated intestinal epithelial cells was detected with antibodies against the extracellular domain of E-cadherin and we demonstrate that this process occurs in a time- and dose-dependent manner. In addition, we showed that the filtered sterile culture supernatant of C. perfringens has no cytotoxic activity on the human intestinal cells at the concentrations used in this study. The direct cleavage of E-cadherin by the proteases from the C. perfringens culture supernatant was confirmed by C. perfringens supernatant-induced in vitro degradation of the human recombinant E-cadherin. We conclude that C. perfringens culture supernatant mediates digestion of epithelial cell junctional proteins which is likely to enable access to the extracellular matrix components by the paracellular pathway.