The involvement of estrogen (E2) and hypoxia in tumor progression is well established. Hypoxia has been reported to activate and degrade estrogen receptor alpha (ERα) in breast cancer cells. Furthermore, E2 has been shown to regulate hypoxia-inducible factor (HIF)-1α protein, but its role in HIF-2α regulation remains largely unexplored. In this study, we found that both HIF-2α mRNA and protein were down-regulated in ER positive but not ER negative breast cancer cells upon treatment with E2. The analysis of 690 samples derived from 608 mixed and 82 triple-negative breast cancer patients revealed that high nuclear HIF-2α tumor levels are associated with a worse prognosis specifically in human epidermal growth factor receptor 2 (HER2) and hormone receptor positive patients. Consistently, ERα/HER2 positive breast cancer cells displayed less pronounced downregulation of HIF-2α by E2. Experiments using a histone deacetylase inhibitor indicate that the E2 mediated decrease in HIF-2α mRNA is due to transcriptional repression. A functional estrogen response element (ERE) was identified in the first intron of the gene encoding HIF-2α (EPAS1), suggesting transcriptional co-repressor recruitment by ERα. Our results demonstrate a novel modulation of HIF-2α in breast cancer cells, explaining the opposing regulation between HIF-1α and HIF-2α in hormone-responsive breast cancer.