Acute myocardial infarction (AMI) remains one of the leading causes of morbidity and mortality worldwide. Ischemia/reperfusion (I/R), the most common consequence of AMI treatment, may induce severe myocardial cell injury, yet the precise mechanism continues to be enigmatic. In the present study, we found that miR-129-5p was significantly downregulated in mouse I/R model. Then, we overexpressed miR-129-5p via intravenous injection of specific miR-129-5p agomir before I/R model establishment. MiR-129-5p overexpression dramatically alleviated myocardial injury in I/R mice as evidenced by reduced lactate dehydrogenase (LDH) activity, malondialdehyde (MDA) content, and infract size. We further detected the effect of miR-129-5p on hypoxia/reoxygenation (H/R)-induced H9C2 cell in vitro. MiR-129-5p overexpression improved H9C2 cell viability and inhibited cell apoptosis induced by H/R, accompanied with decreased LDH activity and MDA content. Besides, luciferase reporter assay indicated that suppressor of cytokine signaling 2 (SOCS2) was a downstream target of miR-129-5p. SOCS2 was upregulated in H/R induced H9C2 cells, and miR-129-5p overexpression suppressed SOCS2 expression at both mRNA and protein levels. In addition, SOCS2 overexpression abolished the protective effects of miR-129-5p on H/R-induced H9C2 cells, concomitant with elevated expression of apoptosis-related cleaved poly-(ADP-ribose) polymerase and cleaved caspase-3. In conclusion, our results demonstrated that miR-129-5p alleviated myocardial injury induced by I/R both in vitro and in vivo, and miR-129-5p/SOCS2 axis is a potential therapeutic target for alleviating myocardial injury in AMI patients after reperfusion.