This study was carried out to evaluate the protective effects of chestnut inner shell extract (CISE) on chronic ethanol-induced oxidative stress in liver. Mice were fed a control liquid diet (Normal-control), liquid diet containing ethanol alone (EtOH+Vehicle), or were administered CISE and ethanol (EtOH+CISE) for 6 weeks. Administration of ethanol induced liver damage with significant increase of plasma GOT, GPT, hepatic triglyceride (TG) and thiobarbituric acid reactive substance (TBARS) levels. By contrast, co-treatment of CISE with ethanol significantly decreased the activities of GOT and GPT in the plasma, and hepatic TG and TBARS levels. Histological observations were consistent with the result obtained from hepatic lipid quantification. Moreover, CISE treatment with ethanol decreased CYP2E1 expression and increased activities of catalase and superoxide dismutase, which were significantly inhibited by treatment with ethanol alone. To determine the active compound of CISE, fractionation of CISE was conducted and scoparone and scopoletin were identified as main compounds. These compounds were also shown to inhibit the ethanol-induced reduction in antioxidant enzyme activity in an in vitro model system. These results suggest that CISE has protective effects against ethanol-induced oxidative damage, possibly by inhibition of lipid accumulation, peroxidation and increase of antioxidant defense system in the liver.