Cardioplegic-induced H/R injury results in cardiomyocytic apoptosis. AMPK has been shown to reduce ER stress and the unfolded protein response (UPR). Whether AMPK activation can attenuate cardiomyocytic apoptosis after cardioplegia-induced H/R injury is unknown. Cardiomyocytes were exposed to simulated ischemia by incubation in a hypoxic chamber with intermittent cold cardioplegia solution infusion at 20-minute intervals and subsequently reoxygenated in a normoxic environment. Various doses of AMPK activators (AICAR or metformin) were given 2 days before H/R injury. The cardiomyocytes were harvested after reoxygenation for subsequent examination. With both AMPK activators, the antiapoptotic genes of ER stress and UPR, the subsequent production of proapoptotic proteins was attenuated, and the antiapoptotic proteins were elevated. The activity of the apoptotic effectors of ER stress was also reduced with AMPK activation. Moreover, TUNEL staining showed that AMPK activation significantly reduced the percentage of apoptotic cardiomyocytes after cardioplegia-induced H/R injury. Our results revealed that AMPK activation during cardioplegia-induced H/R injury attenuates cardiomyocytic apoptosis, via enhancement of antiapoptotic and reduction of proapoptotic responses, resulting from lessening ER stress and the UPR. AMPK activation may serve as a future pharmacological target to reduce H/R injury in the clinical setting.