Abstract: The potential mechanisms for the effects of microgravity on marrow mesenchymal stem cells proliferation were studied. Mouse bone marrow-derived mesenchymal stem cells (BMSCs) were cultured using random positioning machine (RPM) to simulate microgravity (SMG). The effects of SMG on BMSCs proliferation, microfilaments, and key molecule expressions were detected. Under SMG, BMSCs proliferation was inhibited. Meanwhile, BMSCs microfilaments disrupted or abnormally distributed that resulted in cell shape changed. The expression of focal adhesion kinase (FAK) decreased. Akt and ERK1/2 phosphorylation levels, as well as p85β decreased under SMG. The above findings suggest that the inhibiting effects of SMG on BMSCs proliferation are relevant with disrupted cytoskeleton, decreased cell adhesion that interfere with cell growth signal pathway.