Acute pancreatitis (AP) is a common inflammatory disease mediated by damage to acinar cells and subsequent pancreatic inflammation with infiltration of leukocytes. The neuronal guidance protein, netrin-1, has been shown to control leukocyte trafficking and modulate inflammatory responses in several inflammation-based diseases. The present study was aimed toward investigating the effects of netrin-1 in an in vivo model of AP in mice. AP was induced in C57BL/6 mice by administration of two intraperitoneal injections of L-Arginine (4 g/kg). Mice were treated with recombinant mouse netrin-1 at a dose of 1 µg/mouse or vehicle (0.1% BSA) intravenously through the tail vein immediately after the second injection of L-Arginine, and every 24 h thereafter. Mice were sacrificed at several time intervals from 0 to 96 h after the induction of pancreatitis. Blood and tissue samples of pancreas and lung were collected and processed to determine the severity of pancreatitis biochemically and histologically. Immunohistochemical staining demonstrated that netrin-1 was mainly expressed in the islet cells of the normal pancreas and the AP model pancreas, and the pancreatic expression of netrin-1 was down-regulated at both the mRNA and protein levels during the course of AP. Exogenous netrin-1 administration significantly reduced plasma amylase levels, myeloperoxidase activity, pro-inflammatory cytokine production, and pancreas and lung tissue damages. Furthermore, netrin-1 administration did not cause significant inhibition of nuclear factor-kappa B activation in the pancreas of L-Arginine-induced AP. In conclusion, our novel data suggest that netrin-1 is capable of improving damage of pancreas and lung, and exerting anti-inflammatory effects in mice with severe acute pancreatitis. Thus, our results indicate that netrin-1 may constitute a novel target in the management of AP.