Objective To investigate the protective effect of melatonin (Mel) on myocardial injury after myocardial infarction (MI) with diabetes mellitus(DM) by mediating mitophagy. Methods Thirty-two male C57BL/6J mice were randomly divided into four groups, with eight mice in each group, that is, sham operation group (sham group), DM group, MI with DM group (DMI group), and DMI+Mel group. Echocardiography was performed after four weeks to detect the cardiac function, and hearts were removed and sectioned for staining to detect the degree of myocardial interstitial fibrosis and myocardial apoptosis. H9c2 cells were cultured and divided into four groups, that is, control group (CON group), high glucose and high fatty acid group (HG/HF group), HG/HF+hypoxia group (HG/HF+hypoxia group) and HG/HF+hypoxia+Mel group. The apoptosis-related protein levels of B-cell lymphoma-2 (Bcl-2), cysteinyl aspartate specific proteinase (Caspase-3), Bcl2-associated X (Bax) and the mitophapy-related protein levels of microtubules associated protein 1 light chain3-β (LC3), sequestosome-1 (P62), Parkin protein were detected by Western blotting, and the intracellular adenosine triphosphate(ATP) concentration was detected by the luciferase enzyme. Results Compared with the sham group, the left ventricular ejection fraction (LVEF)and left ventricular fractional shortening (LVFS) of DMI group and DMI+Mel group were significantly decreased (P＜0.05), and the left ventricular end-systolic dimension (LVESD), left ventricular end-diastolic dimension (LVEDD), cardiomyocyte cross-sectional area, myocardial interstitial fibrosis area and myocardial apoptosis rates were significantly increased (P＜0.05). Compared with the DMI group, the cardiac function of the DMI+Mel group was significantly improved (P＜0.05), and the LVESD, LVEDD, cardiomyocyte cross-sectional area, myocardial interstitial fibrosis area and myocardial apoptosis rates were significantly decreased (P＜0.05). Compared with the CON group, the expression levels of Bax, Caspase-3, P62 of the HG/HF group and HG/HF+hypoxia group were significantly decreased (P＜0.05), the expression levels of Bcl-2, LC3-Ⅱ/LC3-Ⅰ, Parkin and the intracellular ATP concentration were significantly decreased (P＜0.05). Compared with the HG/HF group and HG/HF+hypoxia group, Mel supplementation can reverse these results. Conclusion Melatonin is likely to reduce myocardial injury after MI with DM by enhancing mitophagy level, thereby improving cardiac function.