Objective To investigate the synergic effects of substrate stiffness and topography on the morphology, proliferation and osteogenic differentiation of rat bone mesenchymal stem cells (rBMSCs). Methods rBMSCs were cultured on polydimethylsiloxane (PDMS) substrates with different stiffness (3.5, 0.27 MPa) and ordered ridges and grooves (width 0.3 or 1.8 μm, depth 0.5 μm) or planar substrate. Inverted fluorescence microscope was used to observe the morphology of rBMSCs. CCK-8 reagent was used to detect the proliferation of rBMSCs. Alkaline phosphatase (ALP) kit was used to detect the ALP activity of rBMSCs. Immunofluorescence technique was used to detect the expression of osteocalcin (OCN) and collagen I (COL Ⅰ). QRT-PCR technique was used to detect the expression of Runx2 mRNA. Results rBMSCs cultured on substrate with 3.5 MPa stiffness, 0.3 μm ridge width, 0.3 μm groove width showed greater proliferation, spreading, cytoskeleton arrangement, and OCN and COL Ⅰ secretion, ALP activity, Runx2 mRNA expression were significantly increased as compared to cells cultured on other groups. Conclusions Substrate stiffness has an obvious influence on rBMSCs proliferation, while substrate stiffness and topography can synergistically promote the proliferation and osteogenic differentiation of rBMSCs. The research findings not only help to understand the biophysical factors in the pathogenesis of certain diseases (such as osteoporosis), but also provide a theoretical basis for developing new materials for bone tissue engineering.