Ischemic preconditioning (IPC) limits myocardial infarct size through the activation of the PI3K–Akt signal cascade; however, little is known about the roles of individual PI3K isoforms in cardioprotection. We aimed, therefore, to elucidate the role of the PI3Kα isoform in cardioprotection Pharmacological PI3Kα inhibition was assessed in isolated-perfused mouse hearts subjected to ischemia/reperfusion injury (IRI), either during the IPC procedure or at reperfusion. PI3Kα inhibition abrogated the IPC-induced protective effect at reperfusion, but not when given only during the IPC protocol. These results were confirmed in an in vivo model. Moreover, pharmacological PI3Kα activation by insulin at reperfusion was sufficient to confer cardioprotection against IRI. In addition, PI3Kα was shown to be expressed and activated in mouse cardiomyocytes, mouse cardiac endothelial cells, as well as in mouse and human heart tissue. Furthermore, PI3Kα was shown to mediate its effect though the inhibition of mitochondrial permeability transition pore opening. In conclusion, PI3Kα activity is required during the early reperfusion phase to reduce myocardial infarct size. This suggests that strategies specifically enhancing the α isoform of PI3K at reperfusion promote tissue salvage and as such, and could provide a direct target for clinical treatment of IRI.