Drug-induced hepatotoxicity is a major problem in drug development, and oxidative stress is known as one of the causes. Superoxide dismutases (SODs) are important antioxidant enzymes against reactive oxygen species (ROS). Mitochondria are the major source of superoxide production, and SOD2 is mainly localized in mitochondria and, with other SODs, plays an important role in scavenging superoxide. Previously, we reported the establishment of an adenovirus vector with short hairpin RNA against rat SOD2 (AdSOD2-shRNA), and applied this to evaluate drug-induced cytotoxicity. In this study, infection of AdSOD2-shRNA to Fisher 344 rats resulted in a significant decrease of SOD2 mRNA, protein expression, and SOD2 enzyme activity to 28%, 35%, and 39%, respectively, 7 days after infection. Serum AST and ALT were significantly increased by single oral administration of acetaminophen (1000 mg/kg) to these SOD2-knockdown rats without fasting compared with the control adenovirus infected groups. Heme oxygenase-1 protein, known to be induced by oxidative stress, was detected in SOD2-knockdown rats administered acetaminophen. In addition, protein carbonyl and lipid peroxidation, also known to be induced by oxidative stress, were significantly increased in SOD2 knockdown rats. This is the first report of a SOD2-knockdown rat model that could be useful to evaluate the drug-induced hepatotoxicity with high sensitivity.