In wild‐type BALB/c mice, i.p. administration of acetaminophen (APAP; 750 mg/kg) induced intrahepatic IFN‐' mRNA expression and a marked increase in serum transaminase levels, leading to acute lethality of ~45%. Histopathological examination showed centrilobular hepatic necrosis with leukocyte infiltration and a large number of apoptotic hepatocytes 10 and 24 h after APAP challenge. mRNA expression of intercellular adhesion molecule 1, vascular cell adhesion molecule 1, interleukin (IL) 1a, IL‐1P, IL‐6, tumor necrosis factor a, monocyte chemoattractant protein 1, macrophage inflammatory protein (MIP) 1a, MIP‐2, KC, IP‐10, Mig, Fas, and inducible nitric oxide synthase was enhanced in the liver of wild‐type mice injected with APAP. To clarify the role of IFN‐' in this process, IFN‐'‐deficient mice were treated in the same manner. All IFN‐'‐deficient mice survived with reduced serum transaminase elevation and attenuated hepatic necrosis, leukocyte infiltration, and hepatocyte apoptosis. The gene expression of all molecules was significantly attenuated in IFN‐'‐deficient mice. Administration of an anti‐IFN‐' neutralizing antibody even 2 or 8 h after APAP challenge to wild‐type mice alleviated APAP‐induced liver injury, and all mice survived. Thus, IFN‐' is responsible for APAP‐induced liver injury by mediating leukocyte infiltration, hepatocyte apoptosis, and NO production as well as cytokine and chemokine production. Moreover, immunoneutralization of IFN‐' may be therapeutically effective for developing APAP‐induced liver injury.—Ishida, Y., Kondo, T., Ohshima, T., Fujiwara, H., Iwakura, Y., Mukaida, N. A pivotal involvement of IFN‐' in the pathogenesis of acetaminophen‐induced acute liver injury. FASEB J. 16, 1227–1236 (2002)