Cytochrome P450 2E1 (CYP2E1) is suggested to play a role in alcoholic liver disease, which includes alcoholic fatty liver, alcoholic hepatitis, and alcoholic cirrhosis. In this study, we investigated whether CYP2E1 plays a role in experimental alcoholic fatty liver in an oral ethanol-feeding model. After 4 weeks of ethanol feeding, macrovesicular fat accumulation and accumulation of triglyceride in liver were observed in wild-type mice but not in CYP2E1-knockout mice. In contrast, free fatty acids (FFAs) were increased in CYP2E1-knockout mice but not in wild-type mice. CYP2E1 was induced by ethanol in wild-type mice, and oxidative stress induced by ethanol was higher in wild-type mice than in CYP2E1-knockout mice. Peroxisome proliferator-activated receptor α (PPARα), a regulator of fatty acid oxidation, was up-regulated in CYP2E1-knockout mice fed ethanol but not in wild-type mice. A PPARα target gene, acyl CoA oxidase, was decreased by ethanol in wild-type but not in CYP2E1-knockout mice. Chlormethiazole, an inhibitor of CYP2E1, lowered macrovesicular fat accumulation, inhibited oxidative stress, and up-regulated PPARα protein level in wild-type mice fed ethanol. The introduction of CYP2E1 to CYP2E1-knockout mice via an adenovirus restored macrovesicular fat accumulation. These results indicate that CYP2E1 contributes to experimental alcoholic fatty liver in this model and suggest that CYP2E1-derived oxidative stress may inhibit oxidation of fatty acids by preventing up-regulation of PPARα by ethanol, resulting in fatty liver.