Oxidative stress is regarded as a major pathogenic factor in acute pancreatitis. Inflammation and apoptosis linked to oxidative stress has been implicated in cerulein-induced pancreatitis as an experimental model of acute pancreatitis. Recently, we found that reactive oxygen species mediate inflammatory cytokine expression and apoptosis of pancreatic acinar cells stimulated with cerulein. Omega-3 fatty acids show antioxidant action in various cells and tissues. In the present study, we investigated whether omega-3 fatty acids inhibit cytokine expression in cerulein-stimulated pancreatic acinar cells and whether omega-3 fatty acids suppress apoptotic cell death in pancreatic acinar cells exposed to hydrogen peroxide. We found that omega-3 fatty acids, such as docosahexaenoic acid (DHA) and α-linolenic acid (ALA), suppressed the expression of inflammatory cytokines (IL-1β, IL-6) and inhibited the activation of transcription factor activator protein-1 in cerulein-stimulated pancreatic acinar cells. DHA and ALA inhibited DNA fragmentation, inhibited the decrease in cell viability, and inhibited the expression of apoptotic genes (p53, Bax, apoptosis-inducing factor) induced by hydrogen peroxide in pancreatic acinar cells. In conclusion, omega-3 fatty acids may be beneficial for preventing oxidative stress-induced pancreatic inflammation and apoptosis by inhibiting inflammatory cytokine and apoptotic gene expression of pancreatic acinar cells.