To investigate the effects of WP1066, an STAT3 inhibitor, on the radiosensitivity of hypoxic U251 human glioma cells induced by cobalt chloride (CoCl2), the viability of U251 cells inhibited by CoCl2 and WP1066 was examined by MTT assay. The mRNA and protein levels of HIF-1α were accessed by RT-PCR and western blot, respectively. The radiosensitivity of U251 cells was detected by clonogenic assay. The data showed that the treatment by 100 µmol•L−1 CoCl2 and 0.5 µmol•L−1 WP1066 for 24 h had no cytotoxic effects on U251 cells (p>0.05). CoCl2 increased the expression of HIF-1α and clonogenic ability of U251 cells exposed to different doses of X-rays (p<0.05). Combined treatment by CoCl2 together with WP1066 suppressed the expression of HIF-1α, thus decreased the clonogenic survival fractions of U251 cells. The study reveals that WP1066 could enhance the radiosensitivity of U251 glioma cells under hypoxia conditions. The mechanism might be related to the downregulation of HIF-1α expression.Cited