Background: Myocardial ischemia-reperfusion injury (MIRI) caused by the reperfusion therapy of myocardial ischemic diseases is a kind of major disease that threatens human health and lives severely. There are lacking of effective therapeutic measures for MIRI. MicroRNAs (miRNAs) are abundant in mammalian species and play a critical role in the initiation, promotion, and progression of MIRI. However, the biological role and molecular mechanism of miRNAs in MIRI are not entirely clear. Methods: We used bioinformatics analysis to uncover the significantly different miRNA by analyzing transcriptome sequencing data from myocardial tissue in the mouse MIRI model. Multiple miRNA-related databases, including miRdb, PicTar, and TargetScan were used to forecast the downstream target genes of the differentially expressed miRNA. Then, the experimental models, including male C57BL/6J mice and HL-1 cell line, were used for subsequent experiments including quantitative real-time polymerase chain reaction analysis, western blot analysis, hematoxylin and eosin staining, flow cytometry, luciferase assay, gene interference, and overexpression. Results: MiR-582-5p was found to be differentially upregulated from the transcriptome sequencing data. The elevated levels of miR-582-5p were verified in MIRI mice and hypoxia/reperfusion (H/R)-induced HL-1 cells. Functional experiments revealed that miR-582-5p promoted apoptosis of H/R-induced HL-1 cells via downregulating cAMP-response element-binding protein 1 (Creb1). The inhibiting action of miR-582-5p inhibitor on H/R-induced apoptosis was partially reversed after Creb1 interference. Conclusions: Collectively, the research findings reported that upregulation of miR-582-5p promoted H/R-induced cardiomyocyte apoptosis by inhibiting Creb1. The potential diagnostic and therapeutic strategies targeting miR-582-5p and Creb1 could be beneficial for the MIRI treatment.