Pancreatitis is a disease for which there are numerous etiologies but no effective treatments. Although the expression of the pancreatitis-associated protein-1 (PAP-1) serves as a marker for the disease, its biological function is unknown. The present study was carried out to determine if PAP-1 performs a protective role against oxidative stress-induced pancreatic cell death. For this purpose, we used cerulein-stimulated pancreatic acinar AR42J cells as an experimental model of acute pancreatitis. First, we demonstrated that PAP-1 gene expression is increased by cerulein in a dose- and time-dependent manner. In parallel, the level of active nuclear factor kappaB (NF-κB) was found to be increased in cells treated with cerulein. To test whether activation of the oxidant-sensitive transcription factor NF-κB is mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the primary source of reactive oxygen species, cerulein-stimulated NADPH oxidase activity was suppressed by using the NADPH oxidase inhibitor diphenyleneiodonium and, separately, by anti-sense oligonucleotides directed against NADPH oxidase subunits p22phox and p47phox. We observed that a decrease in NADPH oxidase activity resulted in decreased NF-κB activation and decreased PAP-1 gene expression. To determine whether the cerulein-induced NF-κB activation involves PAP-1 expression, cells were transfected to overexpress the MAD3 double-point IκBα mutant. In response, NF-κB activation and PAP-1 gene expression were decreased. Lastly, we observed that the cerulein-induced reduction in cell viability and increase in apoptosis are reversed by overexpression of PAP-1 in PAP-1-transfected cells. Taken together, these results support the postulate that PAP-1 inhibits cerulein-induced apoptosis in response to NADPH oxidase-mediated NF-κB activation in pancreatic acinar cells.