ARTICLE ABSTRACTTumor hypoxia is often linked to decreased survival in patients with breast cancer and current therapeutic strategies aim to target the hypoxic response. One way in which this is done is by blocking hypoxia-induced angiogenesis. Antiangiogenic therapies show some therapeutic potential with increased disease-free survival, but these initial promising results are short lived and followed by tumor progression. We hypothesized that this may be due to altered cancer stem cell (CSC) activity resulting from increased tumor hypoxia. We studied the effects of hypoxia on CSC activity, using in vitro mammosphere and holoclone assays as well as in vivo limiting dilution experiments, in 13 patient-derived samples and four cell lines. There was a HIF-1α–dependent CSC increase in ER-α–positive cancers following hypoxic exposure, which was blocked by inhibition of estrogen and Notch signaling. A contrasting decrease in CSC was seen in ER-α–negative cancers. We next developed a xenograft model of cell lines and patient-derived samples to assess the hypoxic CSC response. Varying sizes of xenografts were collected and analyzed for HIF1-α expression and CSC. The same ER-α–dependent contrasting hypoxic-CSC response was seen validating the initial observation. These data suggest that ER-α–positive and negative breast cancer subtypes respond differently to hypoxia and, as a consequence, antiangiogenic therapies will not be suitable for both subgroups. Cancer Res; 73(4); 1420–33. ©2012 AACR.