Trypsin-mediated trypsinogen activation (autoactivation) facilitates digestive zymogen activation in the duodenum, but may precipitate pancreatitis if occurs prematurely in the pancreas. Autoactivation of human cationic trypsinogen is inhibited by a repulsive electrostatic interaction between the unique Asp218 on the surface of cationic trypsin and the conserved tetra-aspartate (Asp19-22) motif in the trypsinogen activation peptide (Nemoda Z, Sahin-Tóth M. J Biol Chem 2005; 280:29645-52). Here we describe that this interaction is regulated by chymotrypsin C (caldecrin), which can specifically cleave the Phe18-Asp19 peptide bond in the trypsinogen activation peptide and remove the N-terminal tripeptide. In contrast, chymotrypsin B, elastase 2A or elastase 3A (proteinase E) are ineffective. Autoactivation of N-terminally truncated cationic trypsinogen is stimulated approximately 3-fold and this effect is dependent on the presence of Asp218. Because chymotrypsinogen C is activated by trypsin, and chymotrypsin C stimulates trypsinogen activation, these reactions establish a positive feed-back mechanism in the digestive enzyme cascade of humans. Furthermore, inappropriate activation of chymotrypsinogen C in the pancreas may contribute to the development of pancreatitis. Consistent with this notion, the pancreatitis-associated mutation A16V in cationic trypsinogen increases the rate of chymotrypsin C-mediated processing of the activation peptide 4-fold and causes accelerated trypsinogen activation in vitro.