CD133-expressing glioma cells play a critical role in tumor recovery after treatment and are resistant to radiotherapy. Herein, we demonstrated that glioblastoma-derived CD133-positive cells (GBM-CD133⁺) are capable of self-renewal and express high levels of embryonic stem cell genes and SirT1 compared to GBM-CD133⁻ cells. To evaluate the role of SirT1 in GBM-CD133⁺, we used a lentiviral vector expressing shRNA to knock-down SirT1 expression (sh-SirT1) in GBM-CD133⁺. Silencing of SirT1 significantly enhanced the sensitivity of GBM-CD133⁺ to radiation and increased the level of radiation-mediated apoptosis. Importantly, knock-down of SirT1 increased the effectiveness of radiotherapy in the inhibition of tumor growth in nude mice transplanted with GBM-CD133⁺. Kaplan–Meier survival analysis indicated that the mean survival rate of GBM-CD133⁺ mice treated with radiotherapy was significantly improved by Sh-SirT1 as well. In sum, these results suggest that SirT1 is a potential target for increasing the sensitivity of GBM and glioblastoma-associated cancer stem cells to radiotherapy.