Abstract Glioblastoma stem cells (GSCs) have been implicated in tumor resistance to radio- and chemotherapy. Proliferation of GSCs reportedly requires calcium influx through T-type calcium channels (Cav3.2). In this study we investigated the expression, function, mechanism of action and therapeutic targeting of Cav3.2 with the FDA approved and repurposed drug Mibefradil in glioblastoma (GBM), and GSCs. We found that Cav3.2 is highly expressed in the majority of human GBM specimens and all GCSs. TCGA data analysis demonstrated that approximately 11% of GBM tumors have upregulated Cav3.2 and that overexpression of Cav3.2 is associated with worse prognosis. Furthermore, we demonstrated that Mibefradil inhibits GSC growth and survival and sensitizes GSCs to Temozolomide (TMZ) chemotherapy. Mibefradil inhibited hypoxia inducible factor HIF1a and induced GSC differentiation. To investigate the mechanism of action of Mibefradil, were performed proteomic and transcriptomic screenings of Mibefradil-treated GSCs using reverse phase protein arrays and RNA-seq, followed by functional rescue experiments. Inhibition of Cav3.2 with Mibefradil significantly altered multiple cancer regulatory signaling pathways and molecules as well as the transcription of oncogenes and tumor suppressors. Among other, Mibefradil suppressed GSC growth through inhibition of pro-survival pathways such as AKT/mTOR, whilst simultaneously inducing apoptosis through upregulation of survivin, BAX and cleavage of caspase 9 and PARP. Notably also, RNA-deep sequencing of Mibefradil treated GSCs revealed an increase in expression of tumor suppressors such as TNFRSF14 and HSD17B14 along with a decrease in the expression of several oncogenes such as PDGFA, PDGFB and TGFB1. We also assessed the therapeutic effects of Mibefradil, on established GSC-derived xenografts. Oral administration of Mibefradil significantly inhibited tumor growth, prolonged animal survival and sensitized tumors to inhibition by TMZ and radiation. This study represents the first comprehensive characterization of Cav3.2 in GBM and GSC. The data establish Cav3.2 inhibition by the repurposed FDA-approved drug Mibefradil as a new strategy for GBM therapy. Citation Format: Ying Zhang, Nichola Cruickshanks, Fang Yuan, Baomin Wang, Mary Pahuski, Julia Wulfkuhle, Isela Gallagher, Alexander F. Koeppel, Sarah Hatef, Christopher Papanicolas, Jeongwu Lee, Eli Bar, David Schiff, Stephen D. Turner, Emanuel Petricoin, Lloyd L. Gray, Roger Abouander. Comprehensive characterization of the role of T-type calcium channels in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3147. doi:10.1158/1538-7445.AM2017-3147