To evaluate the effect of empagliflozin (EMPA) in mitigating microvascular and endothelial damage induced by myocardial ischemia-reperfusion (I/R) injury. Fifteen male C57BL/6J mice were randomized into shamoperated group, I/R group and I/R+EMPA group, and in the latter two groups, myocardial I/R injury was induced by ligating the left anterior descending coronary artery followed by reperfusion for 2 h. EMPA treatment was administered at the daily dose of 10 mg/kg for 7 days. After the treatment, the changes in myocardial microvascular structure of the mice were observed under electron microscopy. In the cell experiment, cultured human coronary artery endothelial cells (HCAECs) were treated with 10 μmol/L EMPA before exposure to hypoxia for 45 min followed normoxic culture for 2 h. Western blotting and immunofluorescence assay were performed to observe fibrin accumulation and endothelial cell protein expressions in the myocardial tissues of the mice and in HCAECs, and RT-qPCR was used to detect the expressions of pro-inflammatory cytokines. Electron microscopy revealed significant myocardial microvascular wall thickening and lumen narrowing in mice with myocardial I/R injury. Fibrin accumulation and ICAM1 expression in the microvessels were more pronounced in I/R group than in the sham-operated and I/R + EMPA groups (P < 0.05). EMPA treatment obviously alleviated microvascular occlusion and microthrombus formation induced by I/R injury. At the cellular level, the protein levels of p-eNOS, Fak, and Src kinases in hypoxic exposure group were significantly lower than those in the control and EMPA treatment groups (P < 0.05). Hypoxic exposure significantly reduced mitochondrial DNA replication and transcription and lowered the expression levels of Cox-Ⅰ and Cox-Ⅱ in HCAECs, and these changes were obviously improved by EMPA treatment (P < 0.05). EMPA can alleviate myocardial I/R injury by maintaining mitochondrial homeostasis to protect the microvascular system.