Glioblastoma multiforme (GBM) is the most common malignancy in central nervous system. A small subpopulation of GBM cells known as GBM stem-like cells (GSLCs) were supposed to be the most malignant cells among GBM cells as they are resistant to multiple therapies including radiotherapy. In this study, we set up two GSLCs cell lines from the two parental U87 and U251 glioma cell lines, and studied the expression of apoptosis-related genes alteration in GSLCs before and after irradiation. We found that one of the receptors of TNF-related apoptosis-inducing ligand (TRAIL), DR5, was dramatically up-regulated in GSLCs after irradiation (IR). Although GSLCs are resistant to both TRAIL and radiation treatment alone, the combined treatment with TRAIL and irradiation achieved maximum killing effect of GSLCs due to inducing the expression of DR5 and inhibiting the expression of cFLIP. Therefore, TRAIL and IR combined treatment would be a simple but practical therapeutic strategy for clinical application.