Ischemic preconditioning (IPC) is a potent cellular protective mechanism whereby brief periods of sublethal ischemia protect the myocardium from prolonged ischemia-induced injury. We demonstrate the selective role of phosphatidylinositol 3-kinase (PI3K) isoforms in IPC. Hearts from PI3Kγ knockout mice (PI3Kγ^−/−) displayed poorer functional recovery and greater tissue injury following IPC compared to wild-type and PI3Kγ^+/− hearts. Examination of the cell-signaling pathways revealed restored phosphorylation levels of Akt and glycogen synthase kinase (GSK)3β in wild-type hearts, which were abolished in PI3Kγ^−/− hearts subjected to IPC. Inhibition of GSK3β by LiCl reversed the loss in protection in PI3Kγ^−/− hearts. In contrast, mice expressing a cardiac-specific kinase-deleted PI3Kα (PI3KαDN) were resistant to injury induced by 30 minutes of ischemia followed by 40 minutes of reperfusion. Furthermore, the resistance of PI3KαDN hearts to ischemia/reperfusion correlated with the persistent expression of p110γ and was blocked by the PI3K inhibitor wortmannin, suggesting the possible enhanced cell signaling through the PI3Kγ pathway. These results demonstrate the importance of the PI3Kγ-Akt-GSK3β signaling pathway in IPC. Selective activation of myocardial PI3Kγ may be an attractive target for the treatment of ischemic heart disease.