Deep-frying oil is a source of free radicals that causes oxidative stress in the body and lead to chronic and degenerative diseases, including non-alcoholic fatty liver (NAFLD). Steatosis, or fatty liver, is one of NAFLD and is a common result of moderate to severe hepatocellular insult. L-Carnitine involves in the metabolism of fat and has a potential role as an antioxidant. In the present study, we aimed to elucidate the role of L-Carnitine in deep-frying oil-treated rats. We equally divided thirty-six male Wistar albino rats into three groups: the group of rats without any treatment (control group), the group of rats treated with deep-frying oil of 0.42 ml/rat/day (negative control group), and the group of rats co-treated with deep-frying oil of 0.42 ml/rat/day and L-Carnitine of 30 mg/kg/day (treatment group). After three weeks of experimental treatment, we found that the deep-frying oil treatment in negative control group caused a significant elevation in the number of hepatic steatosis, hepatic TG content and serum ALT and AST accompanied by increased MDA levels, suggesting the oxidative stress-induced fatty liver. The treatment of L-Carnitine reduced the number of hepatic steatosis and TG content, as well as serum ALT and AST levels. To test the involvement of antioxidant activity of L-Carnitine to its beneficial effects on the development of fatty liver, we observed the expression of endogenous antioxidant. We found that the expression of hepatic SOD, CAT, and GPx were up-regulated by L-Carnitine, followed by a concomitant depletion of MDA levels. In general, this study suggests that L-Carnitine prevents the development of hepatic steatosis and oxidative damage, as well as improves the hepatic antioxidant defenses.