Ischemeia-reperfusion (I/R) injury is a severe complication after restoring blood perfusion in acute myocardial infarction treatment, in which vascular endothelial cell dysfunction is considered as the key event to exacerbate myocardial injury. We have previously verified the protective function of ZNF580 in endothelial cells, however, the impact of ZNF580 on I/R injury and its underlying mechanisms have not been explored in depth. The purpose of the present study is to investigate the regulatory role of ZNF580 on myocardial I/R injury and confirm that ZNF580 is a potential therapeutic candidate for I/R injury treatment. The potential mechanism of ZNF580 in I/R injury was determined via bioinformatics. A model of I/R injury in human umbilical vein endothelial cells (HUVECs) was subsequently established to confirm whether ZNF580 protects against I/R injury and whether this protective effect is exerted through the regulation of autophagic flow. Our study identified 459 differentially expressed genes (DEGs) in I/R injury. ZNF580 increased cell viability and gradually restored cell morphology, the cytoplasm was full, the intracellular structure was clear, and the cell space was significantly reduced in HUVECs exposed to I/R injury. Both Western blotting and reverse transcription-polymerase chain reaction (RT-qPCR) were used to detect the levels of different apoptosis-related proteins, it is shown that the ZNF580 significantly increased lysosome-associated membrane protein 2 (LAMP2) and light chain 3 (LC3) expressions, and markedly decreased protein 62 (P62) expression. Moreover, ZNF580 decreased lactate dehydrogenase (LDH) levels in the supernatant and the rate of apoptosis. ZNF580 promoted autophagosome and lysosome fusion and increased autophagic flux, thereby protecting HUVECs from I/R injury. Its protective effect is possibly related to the activation of the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway.