Tumor cells often develop resistance to radiotherapy by fractionated radiation possibly due to the heterogeneity and hypoxia in tumor tissue. However, the mechanism of refractory effect remains unclear. In the present study, a radioresistant variant HepG2/R60 cell line was isolated from human HepG2 cells by repeated exposure to radiation. The results showed that, after irradiation, the higher survival rate was in HepG2/R60 cells compared to parental cells. Hypoxia treatment could further increase the radioresistance of HepG2/R60 cells concomitant with high level of intracellular GSH and overexpression of HIF-1α. When hypoxic HepG2/R60 cells were pretreated with BSO, a GSH depleter, the refractory response was significantly reduced showing a decrease in intracellular GSH level, followed by the suppression of HIF-1α in hypoxic cells. Subsequent study found that the level of BCL-2 was down-regulated, targeted by HIF-1 prompting transcription in hypoxic cells. The effect of HIF-1α on the radiosensitivity of hypoxic cells was confirmed using YC-1, a specific inhibitor of HIF-1α. Consequently, our results suggest that the radiosensitivity of tumor cells might be regulated by fractionated radiation, and the radioresistance of cells induced by repeated exposure, under hypoxic condition, could be correlated with overexpression of HIF-1α dependent on the alteration of intracellular GSH contents.