We examined the microRNA profiles of Glioblastoma stem (CD133+) and non-stem (CD133-) cell populations and found up-regulation of several miRs in the CD133- cells, including miR-451, miR-486, and miR-425, some of which may be involved in regulation of brain differentiation. Transfection of GBM cells with the above miRs inhibited neurosphere formation and transfection with the mature miR-451 dispersed neurospheres, and inhibited GBM cell growth. Furthermore, transfection of miR-451 combined with Imatinib mesylate treatment had a cooperative effect in dispersal of GBM neurospheres. In addition, we identified a target site for SMAD in the promoter region of miR-451 and showed that SMAD3 and 4 activate such a promoter-luciferase construct. Transfection of SMAD in GBM cells inhibited their growth, suggesting that SMAD may drive GBM stem cells to differentiate to CD133- cells through up-regulation of miR-451 and reduces their tumorigenicity. Identification of additional miRs and target genes that regulate GBM stem cells may provide new potential drugs for therapy.