Hypoxic regions within solid tumors harbor cells that are resistant to standard chemotherapy and radiotherapy. Because oxygen is required to produce ATP by oxidative phosphorylation, under hypoxia, cells rely more on glycolysis to generate ATP and are thereby sensitive to 2-deoxy-d-glucose (2-DG), an inhibitor of this pathway. Universally, cells respond to lowered oxygen tension by increasing the amount of glycolytic enzymes and glucose transporters via the well-characterized hypoxia-inducible factor-1 (HIF). To evaluate the effects of HIF on 2-DG sensitivity, the following three models were used: (a) cells treated with oligomycin to block mitochondrial function in the presence (HIF(+)) or absence (HIF(-)) of hypoxia, (b) cells treated with small interfering RNA specific for HIF-1alpha and control cells cultured under hypoxia, and (c) a mutant cell line unable to initiate the HIF response and its parental HIF(+) counterpart under hypoxic conditions. In all three models, HIF increased resistance to 2-DG and other glycolytic inhibitors but not to other chemotherapeutic agents. Additionally, HIF reduced the effects of 2-DG on glycolysis (as measured by ATP and lactate assays). Because HIF increases glycolytic enzymes, it follows that greater amounts of 2-DG would be required to inhibit glycolysis, thereby leading to increased resistance to it under hypoxia. Indeed, hexokinase, aldolase, and lactate dehydrogenase were found to be increased as a function of HIF under the hypoxic conditions and cell types we used; however, phosphoglucose isomerase was not. Although both hexokinase and phosphoglucose isomerase are known to interact with 2-DG, our findings of increased levels of hexokinase more likely implicate this enzyme in the mechanism of HIF-mediated resistance to 2-DG. Moreover, because 2-DG is now in phase I clinical trials, our results suggest that glycolytic inhibitors may be more effective clinically when combined with agents that inhibit HIF.