Background <p>microRNA-221-3p is highly expressed after myocardial infarction and can target regulate ESR1 expression. This study aims to investigate the effects of MicroRNA-221-3p/ESR1 on hypoxia/reoxygenation(H/R)-induced cardiomyocyte injury.</p> Methods <p>establishment of H/R H9C2 cell model; normal cultured H9C2 cells were designated as the control group. Different treatment methods were applied to H9C2 cells, including miRNA negative control (miR-con), miR-221-3p mimic, miR-221-3p inhibitor, overexpression empty vector (pcDNA-con), and ESR1 overexpression vector (pcDNA-ESR1). The effects of miR-221-3p on cell viability, ROS levels, and the expression of glutathione, 4-hydroxynonenal(4-HNE), Estrogen receptor 1(ESR1), malondialdehyde (MDA) and solute carrier family 7 member 11(SLC7A11) were examined. Mitochondrial membrane potential was assessed using JC-1 staining.</p> Results <p>miR-221-3p is highly expressed in H/R H9C2 cells, and the miR-221-3p mimics further leads to a decrease in cell activity, a decrease in glutathione levels, an increase in 4-HNE, MDA and ROS, and a decrease in the expression of Mitochondrial membrane potential, ESR1 and SLC7A11 genes. The use of miR-221-3p inhibitors or overexpression of ESR1 can partially reverse the damage induced by the miR-221-3p mimic.</p> Conclusions <p>miR-221-3p targets ESR1 to downregulate the protein expression of SLC7A11, causing damage to H/R cardiomyocytes and potentially involving ferroptosis.</p>