Acute myocardial infarction (AMI) rates are rising due to the increasing prevalence of cardiac metabolic disorders, particularly diabetes mellitus (DM). While revascularization procedures such as coronary artery bypass grafting (CABG) and percutaneous coronary intervention (PCI) restore blood flow, they can also exacerbate myocardial ischemia-reperfusion (I/R) injury, for which current cardioprotective strategies remain insufficient. MicroRNAs are critical regulators of gene expression, but the role of miR-100-5p in I/R injury remains unclear. This study demonstrates that miR-100-5p is significantly upregulated in myocardial tissue during I/R injury, leading to the downregulation of protein arginine methyltransferase 5 (PRMT5). This reduction in PRMT5 impairs its capacity to methylate and inactivate Phosphatase and tensin homolog (PTEN), resulting in disruption of the PI3K-AKT signaling pathway, increased cardiomyocyte apoptosis, and aggravated myocardial damage. Using antisense oligomers to inhibit miR-100-5p, we restored PRMT5 expression, reactivated PI3K-AKT signaling, and reduced cardiomyocyte death, thereby mitigating myocardial injury. Our findings identify the miR-100-5p/PRMT5/PI3K-AKT axis as a key regulatory pathway in myocardial I/R injury and highlight miR-100-5p as a potential therapeutic target to protect the heart during revascularization procedures.