Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with poor progrnosis and a high recurrence rate after surgery. To this end, we examined the role of Nanog that is highly expressed in this tumor. NANOG is a transcription factor involved in the pluripotency of embryonic stem cells (ESCs) and the induction of malignancy in cancer stem-like cells (CSCs). Bioinformatic analysis revealed that NANOG may be associated with the development of stem-like traits in GBM. Forced expression of NANOG markedly increased the expression of cancer stem cell markers and promoted the sphere formation and migration of GBM cells. Nanog enhanced the expression of SHH which is required for the maintenance of the positive feedback loop of Hedgehog signaling pathway. Treatment of GBM cells with SANT-1 and GANT61 significantly reduced the tumor progression. These data support a view that reduction of Nanog might have therapeutic benefits in GBM.