Acute pancreatitis (AP) is an inflammatory disease characterized by acinar cell damage, oxidative stress, and inflammation of the pancreas. Nobiletin (3′,4′,5,6,7,8-hexamethoxyflavone), a major polymethoxy flavone, has shown health-promoting properties in previous studies. Therefore, in this study, we investigated whether nobiletin protects against experimental AP induced with l-arginine. C57BL/6 mice were treated with 25 or 50mg/kg nobiletin by intraperitoneal injection once daily for 14 consecutive days. AP was then induced in the mice with two intraperitoneal injections of l-arginine (4g/kg). The nobiletin treatment significantly reduced the plasma amylase levels, pancreatic myeloperoxidase activity, percentage of pancreatic necrosis, plasma proinflammatory factors, the generation of reactive oxygen species, cell apoptosis, tissue damage, and the expression of phosphorylated p38MAPK (p-p38MAPK) and p-AKT. These results suggest that nobiletin is a new therapeutic method for l-arginine-induced AP in mice.