Background: Acute myocardial infarction (AMI) is a serious cardiovascular disease. This study aimed to investigate the diagnostic value of in AMI patients and explore the effects of miR-363-3p on vascular endothelial injury in an AMI rat model. Methods:The Expression of miR-363-3p was measured by quantitative real-time PCR. A receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic value of miR-363-3p in AMI patients. The biomarkers of endothelial injury were estimated using enzyme-linked immunosorbent assays, and the correlation of miR-363-3p with these markers was assessed. AMI rat model was constructed using coronary artery ligation, and the effects of miR-363-3p on endothelial injury and endothelial cell proliferation were analyzed.Results: Serum expression of miR-363-3p was upregulated in the AMI patients compared with healthy controls. The increased serum miR-363-3p serves a candidate diagnostic biomarker of AMI. The correlation analysis indicated that serum miR-363-3p expression was positively correlated with the concentration of endothelial injury biomarkers in AMI patients. Furthermore, the increased endothelial injury biomarkers in AMI rats were all inhibited by the knockdown of miR-363-3p, and the cell proliferation of human umbilical vein endothelial cells was obviously enhanced by the reduction of miR-363-3p. The prediction resultsshown that Kruppel-like factor 2 (KLF2) is a target of miR-363-3p, and their interaction was proved using a luciferase reporter assay.Conclusions: Collectively, overexpression of miR-363-3p acts as a diagnostic biomarker for patients with AMI, and the downregulation of miR-363-3p improves AMI-associated endothelial injury by targeting KLF2, which indicated that miR-363-3p has a potential to develop the treatment of AMI.