Mutations in the activation peptide of human cationic trypsinogen have been found in patients with chronic pancreatitis. Previous biochemical studies demonstrated that mutations p.D19A, p.D22G, and p.K23R strongly stimulate trypsinogen autoactivation. In the present study, we characterized the cell biological effects of these mutants using human embryonic kidney 293T and AR42J rat acinar cells. We found that relative to wild-type trypsinogen, secretion of the mutants from transfected cells was markedly decreased. This apparent secretion defect was completely rescued by inhibition of autoactivation via (1) inclusion of the small molecule trypsin inhibitor benzamidine in the growth medium; or (2) cotransfection with the physiological trypsin inhibitor SPINK1; or (3) by mutation of the catalytic Ser(200) residue in trypsinogen. In contrast, extracellularly added SPINK1 or other nonpermeable proteinaceous trypsin inhibitors did not restore normal secretion of the mutants, indicating that intracellular autoactivation is responsible for the observed secretion loss. Acinar cells expressing the p.D22G mutant detached from the culture plate over time, became terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive, and exhibited elevated levels of the proapoptotic transcription factor CHOP. The observations indicate that activation peptide mutants of human cationic trypsinogen undergo autoactivation intracellularly, which leads to decreased trypsinogen secretion and eventual acinar cell death. The results thus define a novel pathological pathway for parenchymal injury in hereditary chronic pancreatitis.