Substrate wettability and stiffness, two factors impacting cell behaviors simultaneously, have been attracting much attention to elaborate which one dominates. In this study, hydrophilic poly(2-hydroxyethyl methacrylate) brushes were grafted onto the surfaces of poly(dimethylsiloxane) (PDMS) with elastic moduli of 3.66, 101.65 and 214.97 MPa and decreasing water contact angle from 120.4° to 38.5°. Cell behaviors of three cell lines including mBMSCs, ATDC-5, and C28/I2 were then investigated on the hydrophilic and hydrophobic PDMS with different stiffness, respectively. The proliferation of three cell lines was faster on the hydrophilic PDMS than the hydrophobic PDMS, but the stiffness of the hydrophilic or hydrophobic PDMS did not have a significant impact on cell proliferation. The increase of the stiffness enhanced cell migration, the cell spread and the gene expression proportion of extracellular matrix/intercellular adhesion molecules (integrin + FAK/NCAM + N-cadherin) for all three cell lines, but the increase of the wettability showed small enhancement in cell migration, cell spread and gene expression. Moreover, the cartilage-specific gene expression of SOX9 and COL2 downregulated for all three cell lines with the increasing stiffness. The interpretation of the effect of substrate wettability and stiffness on cell behaviors would function as very useful guideline to direct scaffold fabrication.