〔Abstract〕 Objective To investigate the effects of miR-26a-3p on rat myocardial cell(H9c2) injury induced by hypoxia/reoxygenation(H/R) and its mechanism. Methods H9c2 cardiomyocytes in logarithmic growth phase were subjected to hypoxia(1%O2) for 6 h, and reoxygenated at different times(2, 4, 8, 12 h) to establish H/R model cell. Normoxia group was also set up, and cell proliferation activity was detected by cell counting kit-8(CCK-8). The level of lactic dehydrogenase(LDH) in cell supernatant was determined by colorimetry. The expression levels of miR-26a-3p and Survivin mRNA were detected by real-time fluorescence quantitative PCR(qRT-PCR). The expression level of Survivin protein in the cells was detected by Western blot. H9c2 cells were transfected with miR-26a-3p inhibitor and negative control inhibitor NC, Survivin gene siRNA interference plasmid(si-Survivin) and negative control si-NC, followed by H/R intervention. CCK-8 was used to detect cell proliferation in each group. The activity of superoxide dismutase(SOD) and the content of malonaldehyde(MDA) in cell and the level of LDH in supernatant were determined by colorimetry. The apoptosis level of each group was detected by flow cytometry. The protein expression levels of Bcl-2 associated X protein(Bax), B-cell lymphoma-2(Bcl-2), cleaved caspase-3 and Survivin were detected by Western blot. Targeting relationship between miR-26a-3p and Survivin gene was determined by dual luciferase. Results Compared with the normoxia group, proliferative activity, mRNA and protein expression levels of Survivin in H9c2 cells gradually decreased with the extension of reoxygenation time(P<0.05), while LDH and expression level of miR-26a-3p gradually increased(P<0.05). Down-regulating the expression of miR-26a-3p increased proliferative activity, SOD activity, and expression level of Bcl-2 protein in H9c2 cells exposed to H/R(P<0.05), while MDA content, LDH release amount, apoptosis rate, expression levels of Bax and cleaved caspase-3 protein decreased(P<0.05). Survivin deficiency reversed the protective effect of miR-26a-3p inhibitor on H9c2 cells induced by H/R. Dual luciferase reporter gene assay confirmed that Survivin was the target gene of miR-93-5p. Conclusion miR-26a-3p is highly expressed in cardiomyocyte injury induced by H/R. Inhibition of miR-26a-3p expression can inhibit H/R-induced cardiomyocyte apoptosis and oxidative stress by targeted up-regulation of Survivin expression.