Estrogen stimulates breast development during puberty and mammary tumours in adulthood through estrogen receptor α (ERα). These effects are believed to occur via ERa+ luminal cells and not the mammary stem cells (MaSC) which are ERaneg. The ERa+ luminal cells express the stem cell antigen 1 (Sca-1) and we sought to determine if Sca-1 could define an ERa+ subset of EpCAM+/CD24+ CD49fhi MaSCs. Here we show the MaSC population has a distinct Sca-1+ population that is abundant in pre-pubertal mammary glands. The Sca-1+ MaSC have less stem cell markers and less in-vivo repopulating activity than their Sca-1 counterparts but can generate all lineages of mammary epithelial cells. They specifically entered the cell cycle at puberty suggesting they express hormone receptors. We used transcript profiling and immunohistochemistry to show that the they possess ERα transcript and protein and the ERα target gene, progesterone receptor. To determine whether their numbers were modulated by estrogen we assessed their numbers in estrogen estrogen deficient Estrogen receptor alpha knockout mice and Aromatase knockout (ArKO) mice and showed their levels were significantly increased. We treated the ArKO mice with estrogen supplementation and showed that the Sca-1+ MaSC could be directly suppressed by estrogen supplementation. Tamoxifen treatment in WT mice lead to an increase in their numbers confirming the estrogen sensitivity. In conclusion, Sca-1 enriches for an ERα+ve, estrogen-sensitive subpopulation within the CD24+ CD49fhi MaSC population that may likely to be responsible for the hormonal sensitivity of the developing mammary gland.