Abstract: Melatonin exhibits a wide variety of biological effects, including antioxidant and anti-inflammatory functions. Its antioxidant role impedes the etiopathogenesis of pancreatitis, but little is known about the signaling pathway of melatonin in the induction of antioxidant enzymes in acute pancreatitis (AP). The aim of this study was to determine whether melatonin could prevent cerulein-induced AP through nuclear factor erythroid 2-related factor 2 (Nrf2) and curtail inflammation by inhibition of NF-κB. AP was induced by two intraperitoneal (i.p.) injections of cerulein at 2 h intervals (50 μg/kg) in Sprague-Dawley rats. Melatonin (10 or 50 mg/kg/daily, i.p.) was administered 24 h before each injection of cerulein. The rats were killed 12 h after the last injection. Acinar cell degeneration, pancreatic edema, and inflammatory infiltration were significantly different in cerulein- and melatonin-treated rats. Melatonin significantly reduced amylase, lipase, MPO, and MDA levels, and increased antioxidant enzyme activities including SOD and GPx, which were decreased in AP (P < 0.05). Melatonin increased the expression of NQO1, HO-1, and SOD2 when compared with the cerulein-induced AP group (P < 0.05). In addition, melatonin increased Nrf2 expression, and reduced expressions of tumor necrosis factor-alpha, IL-1β, IL-6, IL-8, and iNOS. The elevated nuclear binding of NF-κB in the cerulein-induced pancreatitis group was inhibited by melatonin. These results show that melatonin increases antioxidant enzymes and Nrf2 expression, and limits inflammatory mediators in cerulein-induced AP. It is proposed that melatonin may play an important role in oxidative stress via the Nrf2 pathway in parallel with reduction of inflammation by NF-κB inhibition.