Glioblastoma (GBM) is the most common adult primary tumor of the CNS characterized by rapid growth and diffuse invasiveness into the brain parenchyma. The GBM resistance to chemotherapeutic drugs may be due to the presence of cancer stem cells (CSCs). The CSCs activate the same molecular pathways as healthy stem cells such as WNT, Sonic hedgehog (SHH), and Notch. Mutations or deregulations of those pathways play a key role in the proliferation and differentiation of their surrounding environment, leading to tumorigenesis. Here we investigated the effect of SHH signaling pathway inhibition in human GBM cells by using GANT-61, considering stem cell phenotype, cell proliferation, and cell death. Our results demonstrated that GANT-61 induces apoptosis and autophagy in GBM cells, by increasing the expression of LC3 II and cleaved caspase 3 and 9. Moreover, we observed that SHH signaling plays a crucial role in CSC phenotype maintenance, being also involved in the epithelial-mesenchymal transition (EMT) phenotype. We also noted that SHH pathway modulation can regulate cell proliferation as revealed through the analysis of Ki-67 and c-MYC expressions. We concluded that SHH signaling pathway inhibition may be a promising therapeutic approach to treat patients suffering from GBM refractory to traditional treatments.