Cardiac ischemia-reperfusion injury usually results in acute myocardial infarction (AMI). MiRNAs have been identified as key regulators of AMI. This study was carried out to investigate the effect of miR-27-3p on cardiomyocyte injury in AMI. CCK-8 and flow cytometry assays were used to evaluate cell viability and apoptosis. The expression levels of miR-27-3p, galectin-3, and hypoxia-inducible factor-1α were measured by qRT-PCR. The relationship among miR-27-3p, galectin-3, and HIF-1α was assessed by bioinformatics analysis and luciferase assay. The effects of miR-27-3p and/or galectin-3 and HIF-1α on the inhibition of cell viability and apoptosis induced by H/R were explored. The expression levels of apoptosis-related proteins were determined by Western blot analysis. The expression levels of miR-27-3p were reduced in both ischemia-reperfusion myocardium and HL-1 cells during hypoxia. Overexpression of miR-27-3p reduced I/R-induced myocardial injury, and HIF-1α can reduce this effect. H/R reduced the expression levels of miR-27-3p in HL-1 cardiomyocytes, and HIF1-α reduced this effect, indicating that HIF1-α could regulate the expression of miR-27-3p, and galectin-3 was a target of miR-27-3p. Finally, overexpression of galectin-3 reduced the protective effect of miR-27-3p on cardiomyocyte injury. The expression levels of HIF1-α were increased, and miR-27-3p was downregulated after AMI. HIF-1α promoted myocardial protection by upregulating miR-27-3p, and downregulation of miR-27-3p promoted myocardium cell injury by targeting galectin-3.