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.