Due to its rapid onset and high rates of fatality, acute myocardial infarction (AMI) has long been one of the most fatal diseases among all types of heart diseases. Therefore, intensive research efforts have been focused on understanding AMI's potential pathogenesis to seek effective treatment options. In the present study, 20 peripheral blood samples were collected from patients with AMI, after which reverse transcription-quantitative PCR analysis revealed that microRNA (miR)-124 levels in the peripheral blood of patients with AMI was significantly elevated compared with that in the control group. In vitro, a model using pcDNA3.1-miR-124 transfected human umbilical vein endothelial cells (HUVECs) indicated that overexpression of miR-124 could significantly promote the apoptosis and suppress the proliferation of HUVECs using flow cytometry, TUNEL assay and Cell Counting Kit-8 assays. Based on the present findings, RNA samples of HUVECs overexpressing miR-124 was extracted and sequenced to explore the gene expression profile after miR-124 overexpression. Gene Set Enrichment Analysis (GSEA) analysis revealed that the downregulated genes were mainly enriched in signaling pathways, such as PI3K-AKT, whilst the upregulated genes were mainly enriched in metabolism-related signaling pathways, such as the metabolism of xenobiotics by cytochrome P450 pathway. Additionally, Rideogram software was used to determine the chromosomal localization of the differentially expressed genes. The results demonstrated that they were distributed on all chromosomes except for chromosome Y. In addition, characteristic profiles of the differentially expressed genes caused by miR-124 overexpression were analyzed using Connectivity Map. In total, two medicines, anisomycin and sanguinarine, which function as p38/MAPK signaling agonists that can inhibit angiogenesis, presented with the highest enrichment scores. Together with the GSEA results, which indicated that the differentially expressed genes were mainly enriched in the angiogenesis-inhibiting PI3K/AKT signaling pathway, the present study reported that high expression of miR-124 was negatively associated with patients with AMI, promoting the apoptosis and suppressing the proliferation of vessel endothelial cells.Keywords: PI3K/AKT signal pathway; RNA sequencing; acute myocardial infarction; microRNA-124.