The digestive enzyme chymotrypsin protects the pancreas against pancreatitis by reducing harmful trypsin activity. Genetic deficiency in chymotrypsin increases pancreatitis risk in humans and pancreatitis severity in mice. Pancreatic chymotrypsin is produced in multiple isoforms including chymotrypsin B1, B2, C and chymotrypsin-like protease (CTRL). Here we investigated the role of CTRL in cerulein-induced pancreatitis in mice. Biochemical experiments with recombinant mouse enzymes demonstrated that CTRL cleaved trypsinogens and suppressed trypsin activation. We generated a novel CTRL-deficient strain (Ctrl-KO) using CRISPR-Cas9 genome engineering. Homozygous Ctrl-KO mice expressed no detectable CTRL protein in the pancreas. Remarkably, the total chymotrypsinogen content in Ctrl-KO mice was barely reduced indicating that CTRL is a low-abundance isoform. When given cerulein, Ctrl-KO mice exhibited lower intrapancreatic chymotrypsin activation and a trend for higher trypsin activation, compared with C57BL/6N mice. Despite the altered protease activation, severity of cerulein-induced acute pancreatitis was similar in Ctrl-KO and C57BL/6N mice. We conclude that CTRL is a minor chymotrypsin isoform that plays no significant role in cerulein-induced pancreatitis in mice.