Pathological angiogenesis is a characteristic feature of glioblastoma multiforme (GBM) where the balance between pro-angiogenic and anti-angiogenic factors are shifted towards the pro-angiogenic phenotype. In this study we sought to determine whether angiostatins are expressed by GBM cells and whether their expression along with other related factors [matrix metalloproteinase (MMP)-2, MMP-9, and collagen type I α1 (COLIA1)] are altered by hypoxia and/or correlated with the levels of cancer stem cell marker CD133. Using qRT-PCR, western blotting, and gelatin zymography, we examined the expression of angiostatins, MMP-2, MMP-9, COLIA1 and CD133 in GBM cell lines cultured under aerobic conditions and hypoxia. Expression levels of MMP-2 and MMP-9 were significantly induced by hypoxia. Angiostatins were detected in all GBM cell lines and were increased by hypoxia while the angiostatin isoform of 38-kDa was the most abundant in GBM cells under aerobic and hypoxic conditions. COLIA1 and CD133 were significantly increased in several GBM cell lines under hypoxia. Despite expression and upregulation of anti-angiogenic factors (e.g. angiostatins) in GBM cells, they are overwhelmed by the overexpression of a larger number of angiogenic factors that shift the angiogenic balance towards the pro-angiogenic phenotype. Thus, an exogenous administration of anti-angiogenic factors may be required to improve the treatment of GBM tumors.