Cytoskeletal alterations occur is several cell types including lymphocytes, glial cells, and osteoblasts, during spaceflight and under simulated microgravity (SMG). One of the potential mechanisms of cytoskeletal gravisensitivity of a cell is disruption of extracellular matrix and integrin interactions. Therefore, we investigated the effects of SMG on F-actin cytoskeleton structure, vinculin focal adhesions, expression of some integrin subtypes and cellular adhesion molecules in mesenchymal stem cells (hMSCs) derived from human bone marrow in vitro. Simulated microgravity was produced by RPM (manufactured by Dutch Space, The Netherlands). Culture flasks with MSCs were settled on the inner platform of RPM for 30 minutes, 6, 24, 48 and 120 h. The results have shown that actin cytoskeleton is very fast reorganized even after 30 minutes of simulated microgravity. The number of cells with disrupted actin cytoskeleton steadily increased with the increasing exposure time. However these changes were reversible because the cells partly recovered their F-actin structure after 120-hours-exposure. In addition, we observed vinculin redistribution inside the cells after 6 hours and subsequent terms of exposures. This process was accompanied with increasing of fluorescence intensity of cells. The expression of integrin-alpha2 increased after exposure in RPM. Also we observed decrease in the number of VCAM-1-positive cells and changes in the expression of ICAM-1. Thus, our findings indicate that SMG leads to reversible microfilament alterations of hMSCs and alters adhesion properties of this type of cells.