The purpose of this study is to investigate the influence of mitochondrial respiratory chain complex I inhibition on the radiosensitivity of HepG2 cells. The complex I inhibitor rotenone was used to inhibit complex I activity on HepG2 cells before X-ray irradiation. The cytotoxicity of rotenone was analyzed by MTT assay at various doses. Rotenone induced dissipation of mitochondrial membrane potential and increase of intracellular ROS production were observed. Intracellular ATP production level was determined using luciferin-luciferase assay kit. We further analyzed cell survival and cell cycle distribution of a combined treatment which HepG2 cells underwent 0.5 µM rotenone pretreatment firstly and irradiated with different doses of X-ray radiation afterwards. Our results suggest rotenone pretreatment prior to X-ray irradiation could induce a sensitizing effect on HepG2 cells by enhancing X-ray radiation induced proliferation inhibition and cell apoptosis.