The goal of this study was to determine the effects of hindlimb unloading (HU) on the ex vivo growth and the osteogenic potential of mesenchymal stem cells (MSCs) from the femurs of rats. Microgravity was simulated by 28-day HU in male Sprague-Dawley (SD) rats, and the bone marrow (BM) was collected from hindlimb femurs of HU or control (CTL) rats. MSCs were isolated from BM and cultured for eight passages. Then MSCs at passages 2, 4, and 8 were induced for osteogenesis or adipogenesis. The results revealed that HU decreased the osteogenic potential of MSCs and also decreased the expression of osteoblast gene marker mRNAs in cells induced by osteogenic conditions. Meanwhile, the expression of Runx2 mRNA and the phosphorylation of ERK were also decreased. There were no significant differences of osteoblast gene marker and Runx2 mRNA expression between cells induced from different passages of MSCs in UH rats. Under adipogenic conditions, HU increased both the adipogenic potential of MSCs and the expression of adipocytic gene marker mRNAs in induced cells. HU also increased the expression of PPAR gamma 2 mRNA, but with no effect on the phosphorylation of p38MAPK. The adipogenic potential of MSCs and the expression of adipocytic gene marker mRNAs in induced cells decreased along with cell cultures under normal gravity. This suggests that the normal gravity during in vitro MSC culture and the centrifugal force produced during cell harvest after each passage could decrease the adipogenic potential of MSCs, but could not reverse the effect of HU on the osteogenic potential of MSCs.