Mesenchymal stem cells (MSCs) can differentiate into multiline age cells. Thus, many efforts have been focused on finding effective ways to selectively differentiate the pluripotent cells into defined lineagecommitted progenitor cells for their suitable use in regenerative medicine. Some studies showed that the lineage of MSCs was influenced by the surface topography where the cells were attached, while other studies showed that its lineage was decided by the rigidity of the matrix where the cells interfaced. Our study showed that both the surface topography and rigidity of the matrix simultaneously affected the shapes of MSCs through the alignment and organization of actin cytoskeletons when MSCs were grown on either soft (1 M Pa) or hard (2 ~ 5 GPa) polymers with groove patterns in various dimensions ranging from 500 nm to 50 μm. Cells were mostly aligned and elongated along all the patterns irrespective of the rigidity of the substrate. However, the degree of the elongation was affected by both the matrix rigidity and dimension of the patterns. This is further supported by the results showing that the orientation of alignment of actin cytoskeletons and their organization were affected by both the rigidity and the topography of the substrate.