Accumulating evidence indicates that glioblastoma stem-like cells (GSCs) are key factors in tumour development, recurrence and chemoresistance. The impairment of stemness and the enhancement of differentiation contributes to the weakening of radiation and chemotherapy resistance of GSCs. We previously found that β-elemene was an effective anti-glioblastoma agent and chemosensitizer. In this study, we examined the distribution of CD133(+) cells in human glioblastoma tissues by immunohistochemistry. Following treatment with β-elemene, the formation of GSC spheres was investigated by manual counting, the proliferation of GSCs was measured with a Cell Counting Kit-8 (CCK-8) assay, and the dispersion of GSC spheres was observed with an inverted microscope. GSC spheres were treated with β-elemene, and the expression levels of CD133, ATP-binding cassette subfamily G member 2 (ABCG2) and glial fibrillary acidic protein (GFAP) were examined by western blotting. After treatment with β-elemene, the volumes and weights of GSC xenografts were measured, and the expression of CD133, ABCG2 and GFAP was evaluated through immunohistochemistry analysis. After treatment with β-elemene and temozolomide (TMZ), GSC viability was examined by the CCK-8 assay, and the volumes and weights of xenografts were measured. We found that CD133(+) cells were assembled in some vascular walls and also sparsely distributed in other parts of glioblastoma tissues. β-elemene decreased the formation of GSC spheres, dispersed GSC spheres and inhibited the proliferation of GSCs in vitro and in vivo. In the GSC spheres and xenografts treated with β-elemene, the expression of CD133 and ABCG2 was significantly downregulated, and the expression of GFAP increased. Furthermore, the sensitivity of GSCs to TMZ was enhanced in vitro and in vivo. These results suggest that β-elemene impaired the stemness of GSC spheres, promoted their differentiation and sensitized GSCs to TMZ. β-elemene will hopefully become a valuable agent to enhance the effects of radiotherapy and chemotherapy.