c-Jun N-terminal kinase (JNK) activation promotes hepatocyte death during acetaminophen overdose, a common cause of drug-induced liver failure. While mitogen-activated protein kinase (MAPK) phosphatase (Mkp)-1 is a critical negative regulator of JNK MAPK, little is known about the role of Mkp-1 during hepatotoxicity. In this study, we evaluated the role of Mkp-1 during acute acetaminophen toxicity. Mkp-1+/+ and Mkp-1-/- mice were dosed ip with vehicle or acetaminophen at 300 mg/kg (for mechanistic studies) or 400 mg/kg (for survival studies). Tissues were collected 1-6 hr post 300 mg/kg dosing to assess glutathione levels, organ damage, and MAPK activation. Mkp-1-/- mice exhibited more rapid plasma clearance of acetaminophen than did Mkp-1+/+ mice, indicated by a quicker decline of plasma acetaminophen level. Moreover, Mkp-1-/- mice suffered more severe liver injury, indicated by higher plasma alanine transaminase activity and more extensive centrilobular apoptosis and necrosis. Hepatic JNK activity in Mkp-1-/- mice was higher than in Mkp-1+/+ mice. Finally, Mkp-1-/- mice displayed a lower overall survival rate and shorter median survival time after dosing with 400 mg/kg acetaminophen. The more severe phenotype exhibited by Mkp-1-/- mice indicates that Mkp-1 plays a protective role during acute acetaminophen overdose, potentially through regulation of JNK.