Glioblastoma multiforme (GBM) is an aggressive, incurable type of brain tumor. Brain tumor stem cells (BTSCs) make up a small subset of GBM cells. BTSCs resist standard treatments, initiate recurrence, and pose a significant challenge for GBM treatment. This study examined gene expression and proliferation of GBM cells in vitro to evaluate the effects of 1α,25-dihydroxyvitamin D3 (vitamin D3) treatment. Vitamin D3 is a safe, natural inhibitor of the hedgehog signaling pathway—a mechanism essential to BTSC function. The data in this study demonstrate that the hedgehog signaling pathway is active in both differentiated GBM cell lines and GBM-derived BTSCs. Vitamin D3 reduced GBM cell proliferation, especially in BTSC lines, although the dose at which vitamin D3 was effective varied within the studies. Vitamin D3 also prevented neurosphere formation from a single cell suspension and induced apoptosis in BTSCs, indicating its potential as a therapeutic agent. Because of active vitamin D3's environmental instability, an in vivo model might provide a more complete indication of its anti-tumor effects. Ultimately, vitamin D3 may enhance standard GBM treatments by disabling BTSCs, but further research is needed to determine vitamin D3's role in modulating the growth of GBM cells.