Allicin (2-propene-1-sulfinothioic acid S-2-propenyl ester, diallyl thiosulfinate) is the main biologically active ingredient in garlic. The present study investigated the protective effect of allicin against cardiomyocyte apoptosis that was induced by ischemia in vitro and the potential molecular mechanisms that were involved in this antiapoptotic effect. The results indicated that allicin increased H9c2 cell activity and attenuated the rate of apoptosis that was induced by ischemia/hypoxia. Intracellular calcium concentrations significantly decreased in the allicin-treated groups. Bax expression significantly decreased, and Bcl-2 expression increased in allicin-treated rats. Nitric oxide blockade significantly inhibited these effects. Allicin also increased the activity of SOD and NO release and decreased MDA levels. Allicin significantly increased the expression of eNOS, Nrf2, and HO-1 proteins. Collectively, these findings demonstrate that allicin protects H9c2 cells against apoptosis, and this protective effect appears to occur via eNOS/NO pathway-mediated antioxidant activity.