Apoptosis linked to oxidative stress has been implicated in pancreatitis. NADPH oxidase has been considered as a major source of reactive oxygen species (ROS) during inflammation and apoptosis in pancreatic acinar cells. Recently we demonstrated that NADPH oxidase subunits Nox1, p27phox, p47 phox, and p67phox are constitutively expressed in pancreatic acinar cells and may contribute to apoptosis in pancreatic acinar AR42J cells stimulated with cerulein. The present study aims to investigate the apoptotic mechanism of pancreatic acinar cells stimulated with cerulein by determining whether cerulein induces apoptosis-inducing factor (AIF) expression and whether cerulein-induced expression of AIF is inhibited by transfection with antisense oligonucleotide (AS ODN) of p47phox or p67phox or treatment with a Ca2+ chelator BAPTA-AM. As a result, cerulein induced the expression of apoptotic gene AIF. Transfection with AS ODN of p47phox or p67phox or treatment with BAPTA-AM inhibited cerulein-induced AIF expression in pancreatic acinar AR42J cells. These results demonstrate that NADPH oxidase and calcium have a role in cerulein-induced apoptosis in pancreatic acinar AR42J cells by inducing the expression of AIF. In conclusion, the increase in intracellular Ca2+ and NADPH oxidase activity may be the upstream event of apoptotic gene (AIF) expression, which contributes to cerulein-induced apoptosis in pancreatic acinar AR42 cells.