The glioblastoma (GBM) microenvironment undergoes adaptations to support tumor progression, including a dysregulated extracellular matrix, with altered heparan sulfate (HS) proteoglycans. We investigated N-deacetylase/N-sulfotransferase-1 (NDST1) because NDSTs are initial modifying enzymes of HS biosynthesis and have key roles in designing the HS sulfation pattern. This, in turn governs interactions with growth factors and other biomolecules. We report that NDST1 expression is lower in GBM than in the normal brain, and that patient-derived GBM cells, grown under neural stem cell culture conditions have lower levels of HS than normal astrocytes. Overexpression of NDST1 in GBM cells with low inherent NDST1 levels stimulates cell migration, reduce cell adhesion, induce EMT markers and increase invasion. Conversely, when NDST1 levels were reduced by shRNA in GBM cells, that had higher baseline expression, we find that invasion is reduced, and instead, self-renewal capacity increases alongside elevated stem cell marker expression. Moreover, overexpression of NDST1 changes chromatin accessibility of gene regulatory regions with the capacity to affect transcription factor expression, and pathways that favors cell motility and invasion. Furthermore, NDST1 overexpression results in increased activation of several receptor tyrosine kinases. This study shows that low NDST1 levels support GBM cell stemness, whereas high NDST1 levels endow tumor cells with a motile cell phenotype. We therefore propose that NDST1 is important for regulation of the balance between proliferation and invasive properties in GBM cells.