Background Ischemia-reperfusion (I/R) injury often results in various diseases including myocardial ischemia, accompanying with increasing prevalence and mortality. Emerging literatures highlight the effects of microRNAs (miRNAs) on the heart related diseases but no research reveals the function of miR-134 in myocardial ischemia. Materials and methods Western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were performed to measure the expression levels of miR-134 and nitric oxide synthase 3 (NOS3) expressions in H9c2 cells, as well as anti-apoptotic proteins or pro-apoptotic proteins. Bioinformatic analysis and luciferase reporter assay were utilized to identify the interrelation between miR-134 and NOS3. Flow cytometry assay and cell counting kit-8 were implemented for the detection of cell apoptosis and cell viability, respectively. Results According to the results, we found that hypoxia/reoxygenation (H/R) led to a higher expression level of miR-134 in H9c2 cells than the normal control. Further experiments demonstrated that miR-134 mimic transfection accelerated the speed of cell apoptosis and impaired the cell proliferative prosperity induced by H/R. By contrast, miR-134 inhibitor played a converse role on cell apoptosis and growth. Moreover, our findings also illustrated that miR-134 can target NOS3 directly and simultaneously attenuate the expression of NOS3. The upregulated NOS3 was beneficial against myocardial I/R injury through hindering cell apoptosis and promoting cell proliferation. In addition, miR-134 knockdown recovered the active status of PI3K/Akt pathway that caused by H/R. Conclusion To sum up, this present work put insights into the crucial effects of miR-134/NOS3 axis in myocardial I/R injury, delivering a potential therapeutic technology in future.