The effect of zingiberene, an active sequiterpene from Zingiber officinale Roscoe, Zingiberaceae, against isoproterenol-induced cardiotoxicity in rats was evaluated. The rats were pre-treated with zingiberene (50 mg/kg bw) for the period of 45 days and isoproterenol (20 mg/kg bw) administered to experimental rats for two consecutive days to create cardiotoxicity. Then, the effect of zingiberene on cardiac diagnostic markers (lactate dehydrogenase, creatine kinase-MB isoenzyme and cardiac troponin), cardiac lipid profile, heart lipid peroxidation and antioxidant system (catalase, superoxide dismutase, glutathione peroxidase, reduced glutathione and oxidized glutathione) was evaluated. Isoproterenol administration predominantly altered the levels of triglycerides, cholesterol, phospholipids, free fatty acids and cardiac biomarker enzymes and decreased the antioxidant enzyme activity. On the other hand, pre-treatment with zingiberene significantly (p < 0.05) protected from isoproterenol-induced cardiotoxicity by attenuating hyperlipidemia and oxidative stress. In conclusion, the present study demonstrated that zingiberene ameliorated isoproterenol-induced cardiotoxicity and suggested a cardioprotective role by regenerating the cardiac defense system.