Glioblastoma remains an incurable brain disease due to the prevalence of its recurrence. Considerable evidence suggests that glioma stem-like cells are responsible for glioma relapse after treatment, which commonly involves ionizing radiation. Here, we found that fractionated ionizing radiation (2 Gy/day for 3 days) induced glioma stem-like cell expansion and resistance to anticancer treatment such as cisplatin (50 μM) or taxol (500 nM), or by ionizing radiation (10 Gy) in both glioma cell lines (U87, U373) and patient-derived glioma cells. Of note, concomitant increase of nitric oxide production occurred with the radiation-induced increase of the glioma stem-like cell population through upregulation of inducible nitric oxide synthase (iNOS). In line with this observation, downregulation of iNOS effectively reduced the glioma stem-like cell population and decreased resistance to anticancer treatment. Collectively, our results suggest that targeting iNOS in combination with ionizing radiation might increase the efficacy of radiotherapy for glioma treatment.