Purpose: The purpose of this study is to systematically study the cell-cycle alterations of glioblastoma stem-like cells (GSLCs) after irradiation, possibly enriching the mechanisms of radioresistance of GSLCs.Materials and methods: GSLCs were enriched and identified, and then the radioresistance of GSLCs was validated by analyzing cell survival, cell proliferation, and radiation-induced apoptosis. The discrepancy of the cell-cycle distribution and expression of cell-cycle-related proteins between GSLCs and glioblastoma differentiated cells (GDCs) after irradiation was completely analyzed.Results: The survival fractions and the cell viabilities of GSLCs were significantly higher than those of GDCs after irradiation. Radiation-induced apoptosis was less prominent in GSLCs than in GDCs. After irradiation with high-dose X-rays, the percentages of GDCs in G2/M phase was evidently increased. However, radiation-induced G2/M arrest occurred less frequently in GSLCs, but S-phase arrest occurred in GSLCs after irradiation with 8Gy. Further mechanistic studies showed that the expressions levels of Cdc25c, Cdc2, and CyclinB1 in GSLCs were not apparently changed after irradiation, while those of p-ATM and p-Chk1 were sharply increased after irradiation in GSLCs. The basal level of Cdc25c expression in GSLCs was much higher than that in GDCs.Conclusions: We explored the cell-cycle alterations and cell-cycle-related proteins expression levels in GSLCs after irradiation, providing a novel mechanism of radioresistance of GSLCs.