The poor prognosis of patients with glioblastoma fuels the search for more effective therapeutic compounds. We previously hypothesised that the neuroleptic olanzapine may enhance antineoplastic effects of temozolomide the standard chemotherapeutic agent used in this disease. This study tested this hypothesis. The anti-proliferative effect of olanzapine was examined by MTT assays and cell count analysis. Soft-agar assays were performed to examine colony-forming ability. In addition, the inhibitory effect of olanzapine on the migratory capacity of U87MG and A172 cells was analyzed by Transwell(®) assays. Moreover, staining for annexin V/propidium iodide or carboxyfluorescein succinimidyl ester was performed prior to flow cytometric analysis in order to better understand the subjacent cellular mechanism. Our initial hypothesis that olanzapine may enhance temozolomide's anti-tumor activity could be confirmed in U87MG and A172 glioblastoma cell lines. Moreover, treatment with olanzapine alone resulted in a marked anti-proliferative effect on U87MG, A172 and two glioma stem-like cells with IC50 values ranging from 25 to 79.9 µM. In U87MG cells, anchorage-independent growth was dose-dependently inhibited. In A172 cells, migration was also shown to be inhibited in a dose-dependent manner. In addition, olanzapine was shown to exert a cell line-dependent pleomorphism with respect to the induction of apoptosis, necrosis and/or cytostasis. Our data show that the neuroleptic olanzapine enhances the anti-tumor activity of temozolomide against glioblastoma cell lines. Moreover, this is the first study to show that olanzapine provides on its own anti-cancer activity in glioblastoma and thus may have potential for repurposing.