Background and purpose Chymotrypsin is a serine protease produced by the pancreas and secreted into the lumen of the small intestine, where it digests food proteins. Due to its presence in the gut lumen, we hypothesized that chymotrypsin activity may be found close to epithelial cells and signals to them via Protease-activated receptors (PARs). We deciphered molecular pharmacology mechanisms for chymotrypsin signaling in intestinal epithelial cells. Experimental approaches The presence and activity of chymotrypsin were evaluated by western blot (WB) and enzymatic activity tests in luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N-terminally-tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC-MS analysis. To study the pharmacology of chymotrypsin signals, we investigated calcium signaling and ERK1/2 activation using calcium mobilization assays and WB in CMT93 intestinal epithelial cells. Key results We found that chymotrypsin was present and active in the vicinity of the murine and human colonic epithelium. Molecular pharmacology studies evidenced that chymotrypsin cleaved both PARs receptors. While chymotrypsin activated calcium and ERK1/2 signaling pathways through PAR2, it disarmed PAR1, preventing further activation by its canonical agonist thrombin. CONCLUSION Our work suggests that the function of chymotrypsin in the gut lumen goes well beyond a simple digestive role. Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis.