The Microtubule-Associated Protein Tau is expressed in several cancers, including low-grade gliomas and glioblastomas. We have previously shown that Tau is crucial for the 2D motility of several glioblastoma cell lines, including U87-MG cells. Using an RNA interference (shRNA), we tested if Tau contributed to glioblastoma in vivo tumorigenicity and analyzed its function in a 3D model of multicellular spheroids (MCS). Tau depletion significantly increased median mouse survival in an orthotopic glioblastoma xenograft model. This was accompanied by the inhibition of MCS growth and cell evasion, as well as decreased MCS compactness, implying N-cadherin mislocalization. Intracellular Signaling Array analysis revealed a defective activation of the PI3K/AKT pathway in Tau-depleted cells. Such a defect in PI3K/AKT signaling was responsible for reduced MCS growth and cell evasion, as demonstrated by the inhibition of the pathway in control MCS using LY294002 or Perifosine, which did not significantly affect Tau-depleted MCS. Finally, analysis of the glioblastoma TCGA dataset showed a positive correlation between the amount of phosphorylated Akt-Ser473 and the expression of MAPT RNA encoding Tau, underlining the relevance of our findings in glioblastoma disease. We suggest a role for Tau in glioblastoma by controlling 3D cell organization and functions via the PI3K/AKT signaling axis.