Hypoxic regions are frequent in glioblastoma (GBM), the most common type of malignant adult brain tumor, and increased levels of tumor hypoxia have been associated with worse clinical outcomes. To unmask genes important in hypoxia, we treated GBM neurospheres in hypoxia and identified monocarboxylate transporter-4 (MCT4) as one of the most upregulated genes. To investigate the clinical importance of MCT4 in GBM, we examined clinical outcomes and found that MCT4 overexpression is associated with shorter patient survival. Consistent with this, MCT4 upregulation correlated with the aggressive mesenchymal subset of GBM, and MCT4 downregulation correlated with the less aggressive G-CIMP (Glioma CpG Methylator Phenotype) subset of GBM. Immunohistochemical analysis of tissue microarrays confirmed that MCT4 protein levels were increased in high-grade as compared with lower-grade astrocytomas, further suggesting that MCT4 is a clinically relevant target. To test the requirement for MCT4 in vitro, we transduced neurospheres with lentiviruses encoding short-hairpin RNAs (shRNAs) against MCT4, resulting in growth inhibition of 50-80% under hypoxia in two lines. MCT4 knockdown was associated with a decreased percentage of cells expressing the stem-cell marker CD133 and increased apoptotic fraction. We also found that flow-sorted CD133-positive cells had almost sixfold higher MCT4 levels than CD133-negative cells, suggesting that the stem-like population might have a greater requirement for MCT4. Most importantly, MCT4 silencing also slowed GBM intracranial xenograft growth in vivo. Interestingly, whereas MCT4 is a well-characterized lactate exporter, we found that both intracellular and extracellular lactate levels did not change following MCT4 silencing, suggesting a novel lactate export-independent mechanism for growth inhibition in GBMs. To identify this potential mechanism, we performed microarray analysis on control and shMCT4-expressing neurospheres and found a dramatic reduction in the expression of multiple Hypoxia-Inducible Factor (HIF)-regulated genes following MCT4 knockdown. The overall reduction in HIF transcriptional response was further validated using a hypoxia response element (HRE)-dependent green-fluorescent protein (GFP) reporter line.