Objective：To isolate and culture glioma stem cell-like cells from human glioma U251 cell line，identify the radiosensitivity，and investigate the relationship between signal transducer and activator of transcription 3（STAT3） gene and radiosensitivity of glioma stem cells. Methods：The CD133+ glioma U251 cells were isolated by magnetic bead sorting method，and the spheroidizing ability and radiosensitivity of CD133+ U251 cells were detected. The shRNA silencing expression plasmid and lentiviral over expression plasmid of STAT3 gene were constructed and identified. The U251 cells were transfected with recombinant plasmid，and the relationship between STAT3 expression and radiosensitivity of glioma cells was investigated by flow cytometry and Western blot. Results：The CD133+ U251 cells sorted by magnetic beads had stronger spheroidizing ability. The inhibition rate of CD133+ cells was significantly lower than that of CD133- cells and normal U251 cells after 2，4，6，8 and 10 Gy doses of irradiation（P=0.000），and the apoptosis proportion was lower. CD133+U251 cells transfected with shRNA silencing plasmid of STAT3 gene and lentivirus overexpression plasmid showed that STAT3 protein expression decreased in the silencing group by Western blot. After 2，4，6，8 and 10 Gy doses of irradiation，the STAT3 silencing group had higher cell inhibition rate than the overexpression group（P=0.000），and the cell cycle was inhibited and the apoptosis increased. Conclusion：The CD133+ glioma cells sorted by magnetic beads have the radiation resistance characteristic of stem cell. The glioma cell radiosensitivity can be improved by down regulating the STAT3 expression.