<p>Glioblastoma (GBM) is the most aggressive and common malignant primary brain tumor. Despite advancements in its treatment, patient prognosis remains poor, underscoring an urgent need for novel therapeutic targets and diagnostic markers. The serine synthesis pathway (SSP) plays a crucial role in cellular metabolism and tumor progression, and its upregulation has been implicated in various cancers. However, the significance of the SSP in GBM has not yet been fully understood. In this study, we found that SSP genes, including phosphoglycerate dehydrogenase (<i>PHGDH</i>), phosphoserine aminotransferase (<i>PSAT1</i>), and phosphoserine phosphatase (<i>PSPH</i>), were overexpressed in GBM. Functional studies demonstrated that depletion of SSP genes significantly impaired GBM cell proliferation, colony formation, cell cycle, and survival in vitro as well as tumor growth in vivo. Moreover, SSP gene knockdown disrupted cytoskeletal structure and suppressed GBM cell migration and invasion, correlating with decreased matrix metalloproteinase (MMP)-2 and MMP-9 expression. Pharmacological inhibition of PHGDH using NCT-503 also attenuated GBM cell motility. These findings highlight critical roles of SSP genes in GBM progression and suggest that SSP genes not only serve as potential therapeutic targets, but also valuable diagnostic markers for GBM.</p>