Abstract: To understand the mechanism of cardiovascular dysfunction in the hyperthyroid condition, the role of oxidative stress was examined in rats treated with 3,5,3′-triiodo-l-thyronine (T3). Treatment of rats daily with T3 (8 μg/100 g BW) for 15 days resulted in an increase in heart weight to body weight ratio, which was ameliorated by antioxidants, melatonin (2 mg/100 g BW) or vitamin E (4 mg/100 g BW). Both melatonin and vitamin E also inhibited rises of lipid peroxidation and hydroxyl radical generation and prevented the inhibition of Cu,Zn-superoxide dismutase in the hypertrophic heart. The expression of the glucose transporter, GLUT4, was reduced in response to T3, which was completely restored by melatonin and partially by vitamin E. However, neither antioxidant prevented down regulation of peroxisome proliferator-activated receptor-α in the hyperthyroid heart. Furthermore, the reduced level of myocyte enhancer factor-2, a regulator of GLUT4 transcription was restored completely by melatonin and partially by vitamin E treatment. Glucose uptake in hypertrophic left ventricular cells was also restored by these antioxidants. The expression of B-type natriuretic peptide, a marker of heart failure, was significantly increased by T3 and ameliorated by melatonin or vitamin E treatments. In general, the beneficial effects of melatonin given as a co-treatment with T3 were better than those induced by vitamin E. These data show that melatonin ameliorates hypertrophic growth of the myocardium induced by hyperthyroidism and provide an insight into the mechanism of reactive oxygen species-mediated down regulation of metabolically important genes such as GLUT4 in the heart.