Brain fatty acid binding protein (FABP7; B-FABP) promotes glioblastoma (GBM) cell migration and is associated with tumor infiltration, properties associated with a poor prognosis in GBM patients. FABP7-expressing neural stem-like cells are known to drive tumor migration/infiltration and resistance to treatment. We have previously shown that FABP7's effects on cell migration can be reversed when GBM cells are cultured in medium supplemented with the omega-3 fatty acid, docosahexaenoic acid (DHA). Here, we use super-resolution imaging on patient-derived GBM stem-like cells to examine the importance of FABP7 and its fatty acid ligands in mitigating GBM cell migration. As FABPs are involved in fatty acid transport from membrane to cytosol, we focus on the effect of FABP7 and its ligand DHA on GBM membrane remodeling, as well as FABP7 nanoscale domain formation on GBM membrane. Using quantitative plasma membrane lipid order imaging, we show that FABP7 expression in GBM cells correlates with increased membrane lipid order, with DHA dramatically decreasing lipid order. Using super-resolution stimulated emission depletion (STED) microscopy, we observe non-uniform distribution of FABP7 on the surface of GBM cells, with FABP7 forming punctate nanoscale domains of ∼100 nm in diameter. These nanodomains are particularly enriched at the migrating front of GBM cells. Interestingly, FABP7 nanodomains are disrupted when GBM cells are cultured in DHA-supplemented medium. We demonstrate a tight link between cell migration, a higher membrane lipid order and increased FABP7 nanoscale domains. We propose that DHA-mediated disruption of membrane lipid order and FABP7 nanodomains forms the basis of FABP7/DHA-mediated inhibition of cell migration in GBM.