Glioblastoma (GBM) stem cells (GSCs), which contribute to GBM unfavorable prognosis, show high expression levels of ATP/P2X7 receptors (P2X7R). Here, we reported that cells exposure to 2'(3')-O-(4-benzoylbenzoyl)-ATP (BzATP), a P2X7R agonist, up-regulated the expression of markers associated to epithelial-to-mesenchymal transition (EMT), a process likely contributing to GSC malignancy, and increased GSC migration/invasiveness like the known EMT inducer, Transforming Growth Factor β1 (TGFβ1). These effects were coupled to phosphorylation of SMAD2, a downstream effector in the TGFβ pathway, suggesting its involvement in P2X7R-mediated activity in GSCs. All BzATP effects, including a decrease in the caspase 3/7 activity in GSC medium, were mostly counteracted by the P2X7R antagonist A438079. Finally, BzATP increased the subunit expression of two main human P2X7R splice variants, the full-length P2X7A and the truncated P2X7B, lacking the carboxylic tail, which have different functional properties depending on their arrangement. Since up-regulation of A/B subunits might favor their assembly into a heterotrimeric P2X7R with great sensitivity towards agonists and cell energy support, this is in line with increased EMT markers expression, cell migration/invasion and GSC survival observed following P2X7R stimulation. As in GBM microenvironment extracellular ATP levels may activate P2X7R, our data suggest a P2X7R role in GBM recurrence/invasiveness.