Background: Glioblastoma multiforme (GBM) is the most common, invasive and deadly primary type of malignant brain tumor. The Phosphatidylinositol-3-Kinase/AKT (PI3K/AKT) pathway is highly active in GBM and has been associated with increased survival and resistance to therapy. The aim of this study is to investigate the effects of AKT inhibition in combination with the current standard of care which consists of irradiation and temozolomide (TMZ) on human malignant glioma cells growing adherent and as multicellular spheroids in vitro. Methods: The effects of the allosteric inhibitor MK2206 combined with irradiation and TMZ were assessed on glioma cells growing adherent and as multicellular 3D spheroids. The interaction was studied on proliferation, clonogenic cell survival, cell invasion, -migration and on expression of key proteins in the PI3K-AKT pathway by western blot. Results: A differential effect was found at low- (1 μM) and high dose (10 μM) MK2206. At 1 μM, the inhibitor reduced phosphorylation of Thr308 and Ser473 residues of AKT in both adherent cells and spheroids. Low dose MK2206 delayed spheroid growth and sensitized spheroids to both irradiation and TMZ in a synergistic way (Combination index <0.35). In contrast, neither low nor high dose MK2206 did enhance therapy sensitivity in adherent growing cells. Effective inhibition of invasion and migration was observed only at higher doses of MK2206 (>5 μM). Conclusions: The data show that a 3D spheroid model show different sensitivity to irradiation when combined with AKT inhibition. Thereby we show that MK2206 has potential synergistic efficacy to the current standard of care for glioma patients.