Abstract Background Exercise is beneficial for pathological myocardial dysfunction and heart failure. We previously reported that miR-17-3p contributed to exercise-induced cardiac growth and protected against cardiac ischemia/reperfusion injury. However, if exercise-induced miR-17-3p can prevent pathological myocardial dysfunction is undetermined. Purpose To investigate if exercise-induced miR-17-3p can prevent pathological myocardial dysfunction. Methods The miR-17-3p expression was examined in phenylephrine (PE, 50μmol/L, 48 h) and angiotensin II (Ang II, 1μmol/L, 48 h) treated primary neonatal rat ventricular cardiomyocytes (NRCM), and the myocardium of thoracic aortic constriction (TAC, 4weeks) or angiotensin II (1.3 mg/kg/day, 4weeks) induced cardiac hypertrophy murine model. miR-17-3p transgenic mice were generated and subjected to TAC or Ang II to investigate the effect of miR-17-3p overexpression in attenuating pathological cardiac dysfunction by echocardiography. Cell area and hypertrophic genes were determined by Wheat Germ Agglutinin (WGA) staining and qRT-PCRs. In addition, cell area and hypertrophic genes (ANP, BNP, β-MHC) were determined by immunolabeling and qRT-PCR in NRCM after the transfection of miR-17-3p mimic or inhibitor. Furthermore, functional rescue assays were performed to identify phosphatase and tensin homolog (PTEN) as a target gene of miR-17-3p, and myocyte enhancer factor 2C (MEF2C) as an upstream regulator of miR-17-3p in pathological cardiac hypertrophy. Results The expression of miR-17-3p was significantly decreased in the heart from TAC or Ang II mouse model, and in PE or Ang II-induced cardiomyocyte hypertrophy model. miR-17-3p overexpression mice displayed improved cardiac function and reduced cardiac hypertrophy after TAC or Ang II treatment in vivo. miR-17-3p mimic significantly attenuated PE or Ang II-induced cardiomyocyte hypertrophy in vitro. Based on functional rescue experiments, PTEN was identified as a direct target of miR-17-3p that mediated its protective effects in cardiac hypertrophy. Moreover, MEF2C was identified as a negative upstream regulator of miR-17-3p involved in the control of cardiac hypertrophy. Proposed mechanism of miR-17-3p Conclusion Exercise-induced miR-17-3p can prevent pathological myocardial dysfunction by targeting PTEN. MEF2C was an upstream regulator of miR-17-3p. Targeting MEF2C/miR-17-3p/ PTEN represents a novel therapeutic strategy for pathological myocardial dysfunction. Acknowledgement/Funding The grants from National Natural Science Foundation of China (81722008, 91639101 and 81570362 to JJ Xiao)