Malignant gliomas are currently treated with temozolomide (TMZ), but often exhibit resistance to this agent. CD133(+) cancer stem cells, a population believed to contribute to the tumor's chemoresistance, bear the activation of Notch and Sonic hedgehog (SHH) pathways. In this study, we examined whether inhibition of both pathways enhances the efficacy of TMZ monotherapy in the context of glioma stem cells. Transcriptional analysis of Notch and SHH pathways in CD133(+)-enriched glioma cell populations showed the activity of these pathways. CD133(+) cells were less susceptible to TMZ treatment than the unsorted glioma counterparts. Interestingly, Notch and SHH pathway transcriptional activity in CD133(+) glioma cells was further enhanced by TMZ exposure, which led to NOTCH 1, NCOR2, and GLI1 upregulation (6.64-, 3.73-, and 2.79-fold, respectively) and CFLAR downregulation (4.22-fold). The therapeutic effect of TMZ was enhanced by Notch and SHH pathway pharmacological antagonism with GSI-1 and cyclopamine. More importantly, simultaneous treatment involving TMZ with both of these compounds led to a significant increase in CD133(+) glioma cytotoxicity than treatment with any of these agents alone (P < 0.05). In conclusion, CD133(+) glioma cells overexpress genes involved in Notch and SHH pathways. These pathways contribute to the chemoresistant phenotype of CD133(+) glioma cells, as their antagonism leads to an additive effect when used in combination with TMZ.