Human bone marrow mesenchymal stromal cells (MSCs) with self-renewal and multiple differentiation potentials are considered a possible cell source for tissue engineering and regenerative medicine. However, the limited amount of MSCs in bone marrow and the loss of differentiation capacity following in vitro expansion restrict their practical application. Effective improvement of MSC proliferation is necessary for the clinical application of MSC-based tissue engineering. The effects of estrogen supplements on proliferation and characterizations of human MSCs were investigated at the present study. Supplements of 17-β estradiol (E2) significantly increase the proliferation of human MSCs in vitro. The dose range of E2 to significantly increase MSC proliferation differs in the gender of MSC donor. E2 supplementation in cell proliferation maintains characterizations of MSCs, including cell surface markers, and osteogenic and adipogenic differentiation capacities. These data indicate that estrogen treatment can play an important role in improving human MSCs' expansion in vitro, which will effectively facilitate MSCs' function in the practical application of tissue engineering and regeneration.