Because cells are sensitive to mechanical forces,microgravity might act on stress-dependent cell changes. Regulation of focal adhesions (FAs) and cytoskeletal activity plays a role in cell maintenance, cell movement,and migration. Human MCF-7 cells were exposed to modeled microgravity (MMG) to test the hypothesis that migration responsiveness to microgravity is associated with cytoskeleton and FA anomalies. MMG acts on MCF-7 cells by disorganizing cytoskeleton filaments (microfilaments and microtubules). Microfilaments in MMG did not display their typical radial array. Likewise, microtubules were disrupted in MCF-7 cells within 4 h of initiation of MMG and were partly reestablished by 48 h. FAs generated inmicrogravity were less mature than those established in controls, shown by reduced FAs number and clustering. In parallel, MMG decreased kinases activity (such as FAK,PYK2, and ILK) of FAs in MCF-7 cells. The expression of both integrinbeta1 and integrinbeta4 were downregulated by MMG. We conclude that cytoskeletal alterations and FAs changes in MMG are concomitant with cell invasion and migration retardation. We suggest that reduced migration response in MCF-7 cells following MMG is linked to changes of cytoskeleton and FAs.