Aim To observe effects of resveratrol （Res） preconditioning on miRNA expression signature in rat hearts, and analyse the relationship between Res-mediated cardiac protection and miR-21. Methods The expression profiles of miRNAs in rat hearts after Res preconditioning were detected with a miRNAs microarray chip and the results were confirmed by qRT-PCR. After myocardial ischemia/reperfusion （I/R） injury model was established, myocardial infarct size, percent of myocardial apoptosis and expression of miR-21 were tested. Results miR-21 was one of the most up-regulated miRNAs in hearts by Res preconditioning and its expression was increased more than 2.5 fold compared with the control. The up-regulation of miR-21 was significantly inhibited by antagomiR-21. Cardiac cell apoptosis and myocardial infarct size were significantly decreased by up-regulation of miR-21. Conclusion miRNA signature in rat hearts with Res preconditioning has revealed that multiple miRNAs are regulated by Res. Among them, Res has a protective effect on I/R by reducing cardiac cell apoptosis via miR-21.