MicroRNAs (miRNAs) are noncoding RNAs of 18 to 24 nucleotides that are involved in posttranscriptional regulation of protein expression. Their role in ischemic preconditioning (IPC) is currently unknown. We hypothesized that miRNAs induced after IPC in the heart may create a preconditioned phenotype through upregulating proteins including endothelial nitric oxide synthase (eNOS)/inducible nitric oxide synthase (iNOS) and heat shock protein (HSP)70, which are implicated in the late-phase protection of IPC. miRNAs were extracted from hearts of ICR mice following IPC. The purified miRNAs were injected in vivo into the left ventricular wall of mice, and, 48 hours later, the hearts were subjected to regional ischemia/reperfusion injury by left anterior descending artery ligation for 30 minutes followed by reperfusion for 24 hour. IPC caused no changes in miRNA-23b and miRNA-483 whereas miRNA-1, miRNA-21and miRNA-24 were significantly increased. The IPC-miRNA treatment caused an increase in eNOS mRNA and protein, whereas iNOS was not changed. HSF-1 (heat shock transcription factor 1) and HSP70 were also increased with IPC-miRNA treatment versus control. Moreover, injection of IPC-miRNA protected the hearts against ischemia/reperfusion injury, as shown by a reduction of infarct size as compared with saline or non-IPC miRNA-treated control. We conclude that IPC-induced miRNAs trigger cardioprotection similar to the delayed phase of IPC, possibly through upregulating eNOS, HSP70, and the HSP70 transcription factor HSF-1.