OBJECTIVE MicroRNAs play an important role in regulating myocardial infarction (MI)-induced cardiac injury. MicroRNA-124 (miR-124) plays a vital role in regulating cellular proliferation, differentiation and apoptosis. Although the alteration of miR-124 was confirmed in peripheral blood of MI patients, little is known regarding the biological functions of miR-124 in cardiomyocytes. This study was designed to explore the role of miR-124 in MI and its underlying mechanisms. METHODS Real-time PCR was used to quantify the microRNAs levels. TUNEL and Flow cytometry were performed to measure cell apoptosis. Western blot analysis was employed to detect expression of Bcl-2, Bax, Caspase-3 and STAT3 proteins. RESULTS We revealed that miR-124 was significantly up-regulated in a mice model of MI and in neonatal rat ventricular myocytes (NRVMs) with H2O2 treatment. H2O2 treatment induced cardiomyocyte injury with reduced cell viability and enhanced apoptotic cell death, whereas silencing expression of miR-124 by AMO-124 (antisense inhibitor oligodeoxyribonucleotides) alleviated these deleterious changes. AMO-124 decreased the expression of Bax and cleaved-caspase-3 and upregulated the expression of Bcl-2 in H2O2-treated NRVMs. Besides, AMO-124 improved mitochondrial dysfunction of NRVMs induced by H2O2 treatment. Moreover, antagomir-124 markedly decreased the infarct area and apoptotic cardiomyocytes and improved cardiac function in MI mice. Furthermore, we identified STAT3 as a direct target of miR-124, and downregulation of miR-124 ameliorated the diminished levels of STAT3 and p-STAT3 (Tyr705) in response to H2O2 or MI. STAT3 inhibitor, stattic, was shown to attenuate the elevation of p-STAT3 in NRVMs with AMO-124 transfection. Inhibiting of STAT3 activity by stattic abrogated protective effects of AMO-124 on H2O2-induced cardiomyocytes apoptosis. CONCLUSIONS Taken together, our data demonstrate that downregulation of miR-124 inhibits MI-induced apoptosis through upregulating STAT3, which suggests the therapeutic potential of miR-124 for myocardial infarction.