Aim To investigate the effect and mechanism of lncRNA NEAT1 on hypoxia/reoxygenation induced oxidative stress injury and apoptosis of cardiomyocytes by regulating miR-206. Methods H9c2 cardiomyocytes were cultured in vitro to construct cardiomyocyte hypoxia/reoxygenation model. Real-time quantitative PCR (RT-qPCR) was used to detect the expression of lncRNA NEAT1 and miR-206 after hypoxia/reoxygenation induction. LncRNA NEAT1 small interference RNA (si-lncRNA NEAT1) and miR-206 mimics (miR-206 mimics) were transfected into H9c2 cell, respectively. After hypoxia/reoxygenation induction, the kit was used to detect the content of malondialdehyde (MDA) and reactive oxygen species (ROS) in the cell and the content of lactate dehydrogenase (LDH) in the cell supernatant, MTT assay was used to detect cell activity, flow cytometry was used to detect cell apoptosis, and Western blot was used to detect the expression of cleaved Caspase-3 and cleaved Caspase-9 proteins. The luciferase reporter assay and RT-qPCR were used to verify the targeted binding relationship between lncRNA NEAT1 and miR-206. Results After the induction of hypoxia/reoxygenation, the expression of lncRNA NEAT1 in H9c2 cell was significantly increased, while the expression of miR-206 was significantly decreased (P＜0.05). After transfection with si-lncRNA NEAT1 or miR-206 mimics, the survival rate of H9c2 cell was significantly increased, MDA, ROS contents and LDH activity were significantly reduced, and the expression of cleaved Caspase-3 and cleaved Caspase-9 proteins were significantly reduced, and the apoptosis rate was significantly reduced (P＜0.05). lncRNA NEAT1 targets miR-206 and negatively regulates miR-206 expression. Inhibition of miR-206 partially reversed the effects of silencing lncRNA NEAT1 on oxidative damage and apoptosis of cardiomyocytes induced by hypoxia/reoxygenation (P＜0.05). Conclusion Silencing lncRNA NEAT1 could reduce hypoxia/reoxygenation-induced oxidative stress injury and apoptosis of cardiomyocytes by up-regulating miR-206, and then exert myocardial protection.