Objective To explore the role of miR-221 in the injury induced by hydrogen peroxide (H₂O₂) in rat myocardial cells (H9c2). Methods The viability of H9c2 cell induced by cell different concentrations of H₂O₂ was determined by MTT. The expression of miR-221 was detected by RT-PCR method. The miR-221 inhibitor and negative control were transferred into H9c2 cells by Lipofectamine 2000, then the cells were divided into normal control group, model control group (H₂O₂ group), negative control group (H₂O₂+ negative control group), inhibition group (H₂O₂+miR-221 inhibitor group). The cell viability was measured by MTT assay. Cell apoptosis was detected by acridine orange staining method. The expression of Bcl-2, Bax, phosphatase and tensin homolog deleted on chromosome ten (PTEN, p-protein kinase B (AKT) were assayed by Western Blot. Results 0,25,50,100,200,400 μmol/L H₂O₂ inhibited H9c2 cell activity gradually, of which 200 mol/L inhibition of cell viability moderate, so as a subsequent induction dose. Compared with normal control group, cell viability was decreased (P< 0.01), cell apoptotic rat was increased (P< 0.01), the expression of Bax and PTEN was upregulated (P< 0.01), the expression of Bcl-2 and p-AKT was downregulated (P< 0.01) in model control group and negative control group. Compared with model control group and negative control group, inhibition group proves the contrary. Conclusions Down-expression of miR-221 could significantly inhibit oxidative stress damage in H9c2 cells, which related to regulation of PTEN/AKT signal pathway.