Ischemic preconditioning is the most powerful protective mechanism known against lethal ischemia. Unfortunately the protection lasts for only a few hours. Here we tested the hypothesis that the heart can be kept in preconditioned state for constant protection against ischemia. In this study we chose BMS-191095 (BMS), a highly selective opener of mitochondrial K_ATP channel (mitoK_ATP), BMS (1 mg/kg, i.p.) was administered to rats every 24 hrs till 96 hrs. In other groups, BMS plus wortmannin (WTN, 15 µg/kg, i.p.), an inhibitor of the phosphatidylinositol 3-kinase (PI-3K), or BMS plus 5-hydroxydecanoic acid (5-HD, 5 mg/kg, i.p.), an inhibitor of mitoK_ATP, or BMS plus L-NAME (30 µg/kg, i.p.), an inhibitor of nitric oxide synthase (NOS) were administered to rats. Rats were then subjected to 30 minutes LAD occlusion and 120 minutes reperfusion. Cardiac function, infarct size, pathological changes and apoptosis were assessed at the end of treatments. Saline treated hearts displayed marked contractile dysfunction and underwent pathological changes. BMS treated rats showed significant improvement in cardiac function and infarct size was significantly reduced in BMS treated hearts. However, protection by BMS was abolished by 5-HD, or WTN, or L-NAME. These data demonstrate that hearts can be chronically preconditioned and retain their ability to remain resistant against lethal ischemia and this protection is mediated by activation of mitoK_ATP via nitric oxide (NO) and PI3K/Akt signaling pathways.