Mangiferin is a well-known xanthone extracted from mango leaves (Mangifera indica Linn). Mangiferin is widely distributed in the bark, peel, leaf, seed, stalk, and kernel of mango and higher plants. The pharmacological properties of mangiferin, including its antioxidant, anticancer, antiaging, antiviral, hepatoprotective, analgesic, and immunomodulatory activities, have been described in several studies. We investigated the effect of mangiferin on isoproterenol-induced apoptosis. Experimental heart failure was induced in rats by intraperitoneal administration of isoproterenol (5 mg/kg) for 7 consecutive days. Rats were divided into five groups: group I (sham rats), group II (isoproterenol alone control), group III (isoproterenol + 25 mg/kg mangiferin), group IV (isoproterenol + 50 mg/kg mangiferin), and group V (isoproterenol + 0.0225 mg/kg digitalis as a positive control). Hemodynamic parameters and body weight, heart weight and liver weight, apoptosis induction, and caspase-3, Bax, and Bcl-2 protein levels were measured, and a histopathological analysis of cardiomyocytes was performed. In addition, apoptosis and protein expression of caspase-3, cleaved caspase-3, Bax, and Bcl-2 were measured in cardiac H9c2 cells. Mangiferin supplementation significantly increased heart rate and improved the maximum rate of decrease in left ventricular (LV) pressure, the maximum rate of increase in LV pressure, and LV systolic pressure. Mangiferin reduced inflammatory cell infiltration and the number of broken myocardial fibers, and decreased apoptosis in cardiomyocytes by reducing proteins levels of caspase-3 and Bax and increasing those of Bcl-2. Our findings suggest that mangiferin has a cardioprotective effect against isoproterenol-induced apoptosis in cardiomyocytes.