Myocardial infarction triggers oxidative DNA damage, apoptosis and adverse cardiac remodeling in the heart. Small ubiquitin-like modifier (SUMO) proteins mediate post-translational SUMOylation of the cardiac proteins in response to oxidative stress signals. Upregulation of isoform SUMO2 could attenuate myocardial injury via increasing protein SUMOylation. The present study aimed to discover the identity and cardioprotective activities of SUMOylated proteins. A plasmid vector for expressing N-Strep-SUMO2 protein was generated and introduced into H9c2 rat cardiomyocytes. The SUMOylated proteins were isolated with Strep-Tactin (R) agarose beads and identified by MALDI-TOF-MS technology. As a result, gamma-actin was identified from a predominant protein band of similar to 42 kDa and verified by Western blotting. The roles of SUMO2 and gamma-actin SUMOylation were subsequently determined in a mouse model of myocardial infarction induced by ligating left anterior descending coronary artery and H9c2 cells challenged by hypoxia-reoxygenation. In vitro lentiviral-mediated SUMO2 expression in H9c2 cells were used to explore the role of SUMOylation of gamma-actin. SUMOylation of gamma-actin by SUMO2 was proven to be a new cardioprotective mechanism from the following aspects: 1) SUMO2 overexpression reduced the number of TUNEL positive cells, the levels of 8-OHdG and p-gamma-H2ax while promoted the nuclear deposition of gamma-actin in mouse model and H9c2 cell model of myocardial infarction; 2) SUMO-2 silencing decreased the levels of nuclear gamma-actin and SUMOylation while exacerbated DNA damage; 3) Mutated gamma-actin ((KR)-R-68/(KR)-R-284) void of SUMOylation sites failed to protect cardiomyocytes against hypoxia-reoxygenation challenge. The present study suggested that SUMO2 upregulation promoted DNA damage repair and attenuated myocardial injury via increasing SUMOylation of gamma-actin in the cell nucleus.