Apigenin is a type of flavonoids, which has been demonstrated to protect myocardium against ischemia/reperfusion (I/R) injury. However, the mechanism is still unclear. We hypothesized that the mechanism of cardioprotective action of apigenin on the I/R-induced injury might be caused via B-cell lymphoma (Bcl) signaling pathway. In this study, an in vitro I/R model was replicated on Langendorff-perfused heart and H9c2 cardiomyocytes by anoxia/reoxygenation (A/R) treatment. The recovery of cardiac contractile function, infarct size, lactate dehydrogenase (LDH) and creatine kinase (CK) in the perfusate, the expression and activity of Bcl-2 and caspase-3, and cardiomyocyte apoptosis were measured in the Langendorff heart undergoing A/R injury. In addition, the cell viability, LDH release, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (Δψm), expression of cytochrome c in the cytosol, and cell apoptosis were examined in the culture of H9c2 cardiomyocytes after the A/R. The results showed that apigenin significantly improved rat heart contractile function, reduced LDH release, infarct size and apoptotic rate, upregulated the expression of Bcl-2 and caspase-3, and downregulated the expression of cleaved caspase-3 after the A/R. Moreover, apigenin increased the cell viability and decreased the release of LDH, production of reactive oxygen species, release of mitochondrial cytochrome c into the cytosol, and cell apoptosis in the culture of H9c2 cardiomyocytes after the A/R. In addition, inhibition of Bcl-2 activity by ABT-737 markedly attenuated the protective effect of apigenin on the A/R-induced myocardium injury. Taken together, we firstly demonstrated that the effect of apigenin against A/R injury in cardiomyocytes involves Bcl-2 signal pathway and at least partly depends on its effect of upregulating the expression of Bcl-2.