Mitochondrial oxidative injury can result in many cardiovascular diseases including cardiac ischemia-reperfusion (I/R) injury. This study was designed to investigate whether microRNA-34a (miR-34a) influences cardiac I/R or hypoxia/reoxygenation (H/R) injury by regulating the mitochondrial apoptotic pathway from oxidative injury.In vivo, myocardial infarction size was examined by Evan blue/TTC staining. Apoptosis was assessed by TUNEL assay. Heart function was measured by echocardiography. Lactate dehydrogenase (LDH) and creatine kinase (CK) were evaluated. In vitro, H9c2 cardiomyocytes were exposed to H/R stimulation. Cell viability was assessed by the CCK-8 assay and apoptosis was detected by Annexin V/PI staining. Mitochondrial superoxide, mitochondrial membrane potential (MMP) and ATP production was evaluated by detection kits, and related proteins were detected by western blotting analysis. We observed that the level of miR-34a was significantly upregulated in I/R rats compared to the sham group. Injection of adenovirus inhibiting miR-34a into the left ventricular anterior wall improved heart function and decreased I/R injury. H9c2 cardiomyocytes exposed to H/R stimulation displayed an obvious increase in miR-34a expression. In addition, miR-34a inhibitor alleviated, whereas miR-34a mimic aggravated H/R-induced mitochondrial injury. Bcl-2 was identified as a target gene of miR-34a by dual-luciferase reporter gene detection. Knockdown of Bcl-2 abolished the cardioprotection of the miR-34a inhibitor in H9c2 cells. In summary,our study demonstrates that inhibition of miR-34a exhibits therapeutic potential in treatment of myocardial I/R injury by restraining mitochondrial apoptosis.