MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate protein expression by cleaving or repressing the translation of target mRNAs. miR-125b, one of the neuronal miRNAs, was recently found to be necessary for stem cell fission and for making stem cells insensitive to chemotherapy signals. Temozolomide (TMZ) is a promising chemotherapeutic agent for treating glioblastomas. However, resistance develops quickly and with a high frequency. Given the insensitivity of some glioblastomas to TMZ and the hypothesis that glioma stem cells cause resistance to drug therapy, exploring the functions and mechanisms of miR-125b action on TMZ-treated glioblastoma stem cells would be valuable. In this study, we found that miR-125b-2 is overexpressed in glioblastoma multiforme tissues and the corresponding stem cells (GBMSC); downregulation of miR-125b-2 expression in GBMSC could allow TMZ to induce GBMSC apoptosis. Additionally, the expression of the anti-apoptotic protein Bcl-2 was decreased after the TMZ+miR-125b-2 inhibitor treatment, while the expression of the proapoptotic protein Bax was increased. Further research demonstrated that the induction of apoptosis in GBMSC is also associated with increased cytochrome c release from mitochondria, induction of Apaf-1, activation of caspase-3 and poly-ADP-ribose polymerase (PARP). Taken together, these results suggest that miR-125b-2 overexpression might confer glioblastoma stem cells resistance to TMZ.