Intense neutrophil infiltration into the liver is a characteristic of acetaminophen-induced acute liver injury. Neutrophil elastase is released by neutrophils during inflammation. To elucidate the involvement of neutrophil elastase in acetaminophen-induced liver injury, we investigated the efficacy of a potent and specific neutrophil elastase inhibitor, sivelestat, in mice with acetaminophen-induced acute liver injury. Intraperitoneal administration of 750 mg/kg of acetaminophen caused severe liver damage, such as elevated serum transaminase levels, centrilobular hepatic necrosis, and neutrophil infiltration, with approximately 50% mortality in BALB/c mice within 48 h of administration. However, in mice treated with sivelestat 30 min after the acetaminophen challenge, all mice survived, with reduced serum transaminase elevation and diminished hepatic necrosis. In addition, mice treated with sivelestat had reduced NOS-II expression and hepatic neutrophil infiltration after the acetaminophen challenge. Furthermore, treatment with sivelestat at 3 h after the acetaminophen challenge significantly improved survival. These findings indicate a new clinical application for sivelestat in the treatment of acetaminophen-induced liver failure through mechanisms involving the regulation of neutrophil migration and NO production.