Estrogen stimulates breast development during puberty and mammary tumors in adulthood through estrogen receptor-α (ERα). These effects are proposed to occur via ERα luminal cells and not the mammary stem cells (MaSCs) that are ERα. Since ERα luminal cells express stem cell antigen-1 (SCA-1), we sought to determine if SCA-1 could define an ERα subset of EpCAM/CD24/CD49f MaSCs. We show that the MaSC population has a distinct SCA-1 population that is abundant in pre-pubertal mammary glands. The SCA-1 MaSCs have less stem cell markers and less in vivo repopulating activity than their SCA-1 counterparts. However, they express ERα and specifically enter the cell cycle at puberty. Using estrogen-deficient aromatase knockouts (ArKO), we showed that the SCA-1 MaSC could be directly modulated by estrogen supplementation. Thus, SCA-1 enriches for an ERα, estrogen-sensitive subpopulation within the CD24/CD49f MaSC population that may be responsible for the hormonal sensitivity of the developing mammary gland.