OBJECTIVE This study examines the modifying effects of thymoquinone (TQ), an agent with antioxidant and hypolipidemic properties, on reactive oxygen species, antioxidant activity, and steatosis in livers of hyperlipidemic rabbit. METHODS Oxidative stress was induced in New Zealand White rabbit by a high-cholesterol diet for 8 weeks. Four groups of six animals each were divided into a control group; a high cholesterol group (received 1% cholesterol diet); a high cholesterol/low TQ group (received 1% cholesterol diet plus TQ 10 mg/kg/day), and a high cholesterol/high TQ (20 mg/kg/day) group. Blood samples were taken at the end of the study and examined for fasting serum glucose, insulin, and aminotransaminases. Hepatic tissue samples were examined for malondialdehyde, protein carbonyls, antioxidant enzymes, and steatosis. RESULTS There was severe hepatic steatosis and elevated serum alanine aminotransferase in the high cholesterol group (group II) but not in the high cholesterol and low or high TQ groups (groups III, IV). The hepatic reactive oxygen species activity in the high cholesterol group was significantly higher while the antioxidant enzymes were lower (P<0.05) when compared with the control (group I) or either of the high cholesterol and TQ groups. CONCLUSIONS TQ attenuates hepatic oxidative stress in fatty liver injury induced by high-cholesterol diet in rabbits.