Mitochondrial dysfunction and ferroptosis play crucial roles in myocardial ischemia/reperfusion (I/R) following heart transplantation. Microsomal glutathione s transferase 1 (MGST1) is widely distributed in mitochondria and has a protective effect against ferroptosis, and its involvement in myocardial I/R injury has not yet been elucidated. In this study, donor hearts from C57BL/6 male mice were subjected to 12 h of ex-vivo cold ischemia treatment and transplanted into the abdomen of recipient mice for 24 h of reperfusion. The results showed that MGST1 was significantly down-regulated in the model heart graft tissues, and overexpressing MGST1 effectively alleviated myocardial infarction, inflammation and histological damage of myocardial tissues in the model group. Subsequently, mouse cardiomyocytes HL-1 cells were subjected to oxygen-glucose deprivation/re‑oxygenation (OGD/R) condition, and MGST1 overexpression reduced cell apoptosis and inflammation in OGD/R-induced HL-1 cells. Of note, MGST1 overexpression attenuated I/R-induced mitochondrial damage and inhibited ferroptosis in vitro and in vivo. Moreover, MGST1 was found to be negatively regulated by DNA methyltransferase 1 (DNMT1)-mediated promoter methylation, and DNMT1 silence suppressed OGD/R-induced damage in HL-1 cells through restoring MGST1 expression. Altogether, targeting MGST1 hyper-methylation ameliorates mitochondrial damage and ferroptosis of cardiomyocytes, and prevents myocardial I/R injury following heart transplantation.