In this study, the PDMS (polydimethyl siloxane) membranes with different mechanical strength were prepared and applied for the culture of osteoblastic cells. The osteoblastic phenotypes were then analyzed, including attachment, proliferation and differentiation. Meanwhile, the hydrophilicity of membranes was also adjusted by using plasma treatment. From the preliminarily results, the osteoblastic cells would prefer to attach to the PDMS substrate with higher hardness, which also resulted in a higher cell density in cell proliferation for longer culture times. The above tendency would be only significant on the hydrophilic surface, which revealed that the cell would not well recognize the surface properties on the hydrophobic surface. The results also indicated the osteoblastic differentiation would be affected by the mechanical strength of substrate surfaces. To investigate the mechanism of the mechanical effects on cell behaviors, the protein deposition was analyzed on surfaces with different hardness. The outcome suggested that the protein composition on these surfaces would be changed due to the difference in the mechanical properties.