The hypoxia-inducible transcription factors (HIF) 1α and HIF-2α play a critical role in cellular response to hypoxia. Elevated HIF-α expression correlates with poor patient survival in a large number of cancers. Recent evidence suggests that HIF-2α appears to be preferentially expressed in neuronal tumor cells that exhibit cancer stem cell characteristics. These observations suggest that expression of HIF-1α and HIF-2α is differentially regulated in the hypoxic tumor microenvironment. However, the underlying mechanisms remain to be fully investigated. In this study, we investigated the transcriptional regulation of HIF-1α and HIF-2α under different physiologically relevant hypoxic conditions. We found that transcription of HIF-2α was consistently increased by hypoxia, whereas transcription of HIF-1α showed variable levels of repression. Mechanistically, differential regulation of HIF-α transcription involved hypoxia-induced changes in acetylation of core histones H3 and H4 associated with the proximal promoters of the HIF-1α or HIF-2α gene. We also found that, although highly stable under acute hypoxia, HIF-1α and HIF-2α proteins become destabilized under chronic hypoxia. Our results have thus provided new mechanistic insights into the differential regulation of HIF-1α and HIF-2α by the hypoxic tumor microenvironment. These findings also suggest an important role of HIF-2α in the regulation of tumor progression under chronic hypoxia.