Rapamycin (sirolimus) is an antibiotic that inhibits protein synthesis through mammalian target of rapamycin (mTOR) signaling and is used as an immunosuppressant in the treatment of organ rejection in transplant recipients. Recently, the antigrowth properties of rapamycin have been utilized for cardiovascular benefit as stents impregnated with rapamycin effectively reduce coronary restenosis. We report here a novel role of this drug in protection against ischemia/reperfusion (I/R) injury. Adult male ICR mice were treated with rapamycin (0.25 mg/kg, IP) or volume-matched DMSO (solvent for rapamycin). The hearts were subjected to 20 min of global ischemia and 30 min of reperfusion in Langendorff mode. The blocker of mitochondrial K_ATP channel, 5-hydroxydecanoate (5-HD, 100 μM) was given 10 min before ischemia. Infarct size in the DMSO treated group was 28.2 ± 1.3% and was reduced to 10.1 ± 2.8% in the rapamycin-treated mice (64% decrease, P <0.001). 5-HD blocked the protective effect (infarct area 32.2 ± 1.8%, P <0.001 vs. rapamycin). The infarct limiting effect of rapamycin was not associated with improved recovery of ventricular function. We further examined the effect of rapamycin in protection against necrosis and apoptosis in adult cardiomyocytes subjected to simulated ischemia and reoxygenation. Myocytes treated with rapamycin in doses from 25–100 nM demonstrated significantly lower trypan blue-positive necrotic cells and TUNEL-positive apoptotic nuclei, supporting the protective role of drug in the intact heart. These data suggest that rapamycin induces potent preconditioning-like effect against myocardial infarction through opening of mitochondrial K_ATP channels. We propose that rapamycin may be a novel therapeutic strategy to limit infarction, apoptosis, and remodeling following I/R injury in the heart.