Oxygen and nutrient limitation are common features of the tumor microenvironment and are associated with cancer progression and induction of metastasis. The inefficient vascularization of tumor tissue also limits the penetration of other serum-derived factors, such as lipids and lipoproteins, which can be rate limiting for cell proliferation and survival. Here we have investigated the effect of hypoxia and serum deprivation on sterol regulatory element-binding protein (SREBP) activity and the expression of lipid metabolism genes in human glioblastoma multiforme (GBM) cancer cells. We found that SREBP transcriptional activity was induced by serum depletion both in normoxic and hypoxic cells and that activation of SREBP was required to maintain the expression of fatty acid and cholesterol metabolism genes under hypoxic conditions. Moreover, expression of stearoyl-CoA desaturase, the enzyme required for the generation of mono-unsaturated fatty acids, and fatty acid-binding protein 7, a regulator of glioma stem cell function, was strongly dependent on SREBP function. Inhibition of SREBP function blocked lipid biosynthesis in hypoxic cancer cells and impaired cell survival under hypoxia and in a three-dimensional spheroid model. Finally, gene expression analysis revealed that SREBP defines a gene signature that is associated with poor survival in glioblastoma.