We investigated the role of microRNAs (miRNA) in protection against ischemia/reperfusion (I/R) injury in heart. Mice subjected to cytoprotective heat-shock (HS) showed a significant increase of miRNA-1, miRNA-21 and miRNA-24 in the heart. miRNAs isolated from HS mice and injected into non-HS mice significantly reduced infarct size after I/R injury, which was associated with the inhibition of pro-apoptotic genes and increase in anti-apoptotic genes. Chemically synthesized miRNA-21 also reduced infarct size, whereas a miRNA-21 inhibitor abolished this effect. Overall, these studies for the first time provide evidence for the potential role of endogenously synthesized miRNA's in cardioprotection following I/R injury.