Objective: Myocardial infarction (MI) remains the leading cause of morbidity and mortality due partly to the limited regenerative capacity of cardiomyocytes to replace cardiomyocyte lost due to apoptosis. Inhibiting cardiomyocyte apoptosis is recognized as an effective therapeutic approach for MI. MicroRNAs (miRNAs, miRs), which regulate target genes at the post-transcriptional level, play a significant role in the regulation of cardiovascular diseases such as MI. MicroRNA-135b (miR-135b) has a protective effect on cardiomyocytes. However, the role of miR- 135b in cardiomyocyte apoptosis in infarct myocardium needs further clarification. Methods: We generated α-MHC-miR-135b transgenic mice to investigate the role of miR-135b in myocardial injury after MI. MiR- 135b mimic and negative control (NC) were transfected into H₂O₂-induced cardiomyocytes to evaluate the effect of overexpression of miR-135b on the levels of reactive oxygen species (ROS) and apoptosis. Results: Our results showed that overexpression of miR-135b had protective effect on cardiomyocyte injury both in vivo and in vitro. MiR-135b inhibited cardiomyocyte apoptosis and ROS generation, downregulated proapoptosis proteins (cleaved-caspase-3 and Bax), and increased antiapoptosis protein (Bcl-2). Moreover, miR-135b showed an inhibitory effect on apoptosis-related protein target transient receptor potential vanilloid-type 4 (TRPV4) cation channel. Conclusion: MiR-135b might be considered a new molecular target for potential replacement therapy as antiapoptotic cardioprotection in the setting of MI.