Calcium (Ca²⁺) signaling is an important signaling process, implicated in cancer cell proliferation and motility of the deadly glioblastomas that aggressively invade neighboring brain tissue. We have previously demonstrated that caffeine blocks glioblastoma invasion and extends survival by inhibiting Ca²⁺ release channel inositol 1,4,5-trisphosphate receptor (IP₃R) subtype 3. Trifluoperazine (TFP) is an FDA-approved antipsychotic drug for schizophrenia. Interestingly, TFP has been recently reported to show a strong anticancer effect on lung cancer, hepatocellular carcinoma, and T-cell lymphoma. However, the possible anticancer effect of TFP on glioblastoma has not been tested. Here, we report that TFP potently suppresses proliferation, motility, and invasion of glioblastoma cells in vitro, and tumor growth in in vivo xenograft mouse model. Unlike caffeine, TFP triggers massive and irreversible release of Ca²⁺ from intracellular stores by IP₃R subtype 1 and 2 by directly interacting at the TFP-binding site of a Ca²⁺-binding protein, calmodulin subtype 2 (CaM2). TFP binding to CaM2 causes a dissociation of CaM2 from IP₃R and subsequent opening of IP₃R. Compared with the control neural stem cells, various glioblastoma cell lines showed enhanced expression of CaM2 and thus enhanced sensitivity to TFP. On the basis of these findings, we propose TFP as a potential therapeutic drug for glioblastoma by aberrantly and irreversibly increasing Ca²⁺ in glioblastoma cells.