A series of novel artemisinin (ART) derivatives containing an isothiocyanate (ITC) group were synthesized. All the compounds showed more potent anti-tumor effects than those of parent dihydroartemisinin (DHA) towards glioblastoma multiforme U87 in vitro. Among them, 5b had the strongest cytotoxic activity which exerted its effects in a concentration-dependent but not time-dependent manner (IC50 7.41 M for 24 h, 7.35 M for 72 h). Pyknosis was observed in 5b-treated U87 cells. Multiple intrinsic apoptotic pathways were induced by 5b including the upregulation of caspase 9, the release of cytochrome c, an increase of the proapoptotic protein Bax, a decrease of the anti-apoptotic protein Bcl 2, and the activation of execution pathways by the upregulation of caspase 3. In addition to apoptosis, an autophagic mechanism was also involved in 5b-induced cytotoxicity to human GBM U87 cells by upregulating the expression of LC3-II and downregulating p62. Furthermore, 5b also significantly attenuated the migration of U87 cells. Therefore, our results suggest that 5b may be a promising molecule for the further development of a novel drug for the treatment of glioblastoma.