The size of the satellite cell pool is reduced in estradiol (E)-deficient female mice and humans. Here, we use a combination of in vivo and in vitro approaches to identify mechanisms, whereby E deficiency impairs satellite cell maintenance. By measuring satellite cell numbers in mice at several early time points postovariectomy (Ovx), we determine that satellite cell numbers decline by 33% between 10 and 14 days post-Ovx in tibialis anterior and gastrocnemius muscles. At 14 days post-Ovx, we demonstrate that satellite cells have a reduced propensity to transition from G/G to S and G/M phases, compared with cells from ovary-intact mice, associated with changes in two key satellite cell cycle regulators, and . Further, freshly isolated satellite cells treated with E in vitro have 62% greater cell proliferation and require less time to complete the first division. Using clonal and differentiation assays, we measured 69% larger satellite cell colonies and enhanced satellite cell-derived myoblast differentiation with E treatment compared with vehicle-treated cells. Together, these results identify a novel mechanism for preservation of the satellite cell pool by E via promotion of satellite cell cycling.