BACKGROUND: The sodium pump α3 subunit (ATP1A3) is associated with various brain's physiological and pathological mechanisms. However, its molecular mechanisms and cellular targets in glioblastoma (GBM) are poorly understood. METHODS: Bioinformatics and phosphor-proteomics analysis, target fishing experiment, confocal immunofluorescence, molecular cloning, and western blot techniques were carried out to elucidate probable downstream signaling pathways. Then GBM xenografts were established to assess potential molecular mechanisms of ATP1A3 associated with its in vivo anti-glioma impacts. RESULTS: The mechanistic analyses indicated that the antagonism between ATP1A3 and small nuclear ribonucleoprotein polypeptide G (SNRPG) could suppress GBM growth. ATP1A3 inhibits SNRPGinduced GBM epithelial-mesenchymal transition, and SNRPG decreases ATP1A3 by increasing phosphorylation at S643. As a negative feedback loop, ATP1A3 overexpression causes a reduction of SNRPG-induced invasion-metastasis cascades via regulating KLF9. Furthermore, by using artificial intelligence (AI) techniques, we have also exerted the design and application of a synthetic peptide (ATP1A3-S643 peptide), which could be the potential inhibitor of ATP1A3 phosphorylation. To better explore the anti-glioma effect of ATP1A3 activation, a bioengineering nanomedicine capable of ondemand ATP1A3 activator delivery to the brain for GBM has also been developed in this work, which exhibited an improved therapeutic efficacy in the ATP1A3-targeted treatment of glioma. CONCLUSION: ATP1A3 is a potential anti-glioma treatment target, and its activation critically depends on its antagonizing interaction with SNRPG.