Malignant gliomas are characterized by a short median survival which is largely impacted by the resistance of these tumors tochemo‐ and radiotherapy. Recent studies suggest that a small subpopulation of cancer stem cells, which are highly resistant to γ‐radiation, has the capacity to repopulate the tumors and contribute to their malignant progression. γ‐radiation activates the process of autophagy and inhibition of this process increases the radiosensitivity of glioma cells; however, the role of autophagy in the resistance of glioma stem cells (GSCs) to radiation has not been yet reported. In this study we examined the induction of autophagy by γ‐radiation in CD133+ GSCs. Irradiation of CD133+ cells induced autophagy within 24–48 hr and slightly decreased the viability of the cells. γ‐radiation induced a larger degree of autophagy in the CD133+ cells as compared with CD133− cells and the CD133+ cells expressed higher levels of the autophagy‐related proteins LC3, ATG5 and ATG12. The autophagy inhibitor bafilomycin A1 and silencing of ATG5 and beclin1 sensitized the CD133+ cells to γ‐radiation and significantly decreased the viability of the irradiated cells and their ability to form neurospheres. Collectively, these results indicate that the induction of autophagy contributes to the radioresistance of these cells and autophagy inhibitors may be employed to increase the sensitivity of CD133+ GSCs to γ‐radiation. © 2009 UICC