Protease-activated receptor 2 (PAR(2)) is a central regulator of intestinal barrier function, inflammation and pain. Upregulated intestinal proteolysis and PAR(2)-signaling are implicated in inflammatory bowel diseases (IBDs) and irritable bowel syndrome (IBS). To identify potential bacterial regulators of PAR(2) activity, we developed a functional assay for PAR(2) processing and used it to screen conditioned media from a library of diverse gut commensal microbes. We found that multiple bacteria secrete proteases that cleave host PAR(2). Using chemoproteomic profiling with a covalent irreversible inhibitor, we identified a previously uncharacterized Bacteroides fragilis serine protease Bfp1, and showed that it cleaves and activates PAR(2) in multicellular and murine models. PAR(2) cleavage by Bfp1 disrupts the intestinal barrier, sensitizes nociceptors, and triggers colonic inflammation and abdominal pain. Collectively, our findings uncover Bfp1-mediated PAR(2)-processing as a new axis of host-commensal-interaction in the gut that has the potential to be targeted for therapeutic intervention in IBD or IBS.