〔Abstract〕 Objective To investigate the effect of sirtuin 3(SIRT3) on the oxidative stress response and hypoxia inducible factor-1α(HIF-1α) expression in lung cancer cells through reactive oxygen species(ROS) under hypoxic conditions and its mechanism. Methods Human non-small cell lung cancer A549 cells were exposed to hypoxia for 0 h, 12 h, 24 h and 48 h. The mRNA and protein expressions of HIF-1α and SIRT3 were detected by RT-PCRand Western blot to determine the optimal time of hypoxia induction. A549 cells were divided into 5 groups: control group, hypoxia group, hypoxia+ROS inhibitor n-acetylcysteine(NAC) group, hypoxia+(SIRT3 overexpression) SIRT3-OE group and hypoxia+SIRT3-OE+NAC group. Cell proliferation was detected by MTT assay. Cell apoptosis was detected by flow cytometry. The mRNA and protein expressions of HIF-1α and SIRT3 in each group were detected by RT-PCR and Western blot. ROS content in each group was detected by flow cytometry. The contents of malondialdehyde(MDA), superoxide dismutase(SOD) and glutathione(GSH) in the cells of each group were detected by biochemical kits. Results The optimal induction time of hypoxia was 24 h. Compared with the control group, the apoptosis rate, SIRT3 mRNA and protein levels, SOD and GSH contents in the hypoxia group significantly decreased(P<0.01), the cell proliferation ability, HIF-1α mRNA and protein levels, ROS and MDA content in cells significantly increased(P<0.01). Compared with the hypoxia group, the apoptosis rate, SIRT3 mRNA and protein levels, SOD and GSH contents in the hypoxia+NAC and hypoxia+SIRT3-OE groups increased(P<0.05), the cell proliferation ability, HIF-1α mRNA and protein levels, ROS and MDA content in cells decreased(P<0.05). Compared with the hypoxia+NAC group, the apoptosis rate, SIRT3 mRNA and protein levels, SOD and GSH contents in the hypoxia+SIRT3-OE+NAC group significantly increased(P<0.01), the cell proliferation ability, HIF-1α mRNA and protein levels, ROS and MDA content in cells significantly decreased(P<0.01). Conclusion Under hypoxic conditions, SIRT3 can promote cell apoptosis and inhibit lung cancer progression by mediating ROS to inhibit oxidative stress response and HIF-1α expression in lung cancer cells.