Fibronectin (Fn) is significant to the performance of biomaterials, and the chemistry of biomaterial surface play important roles in Fn adsorption and subsequent cell behavior. However, the “molecular scale” mechanism is still unclear. Herein, we combined experimental strategies with molecular simulations to solve this problem. We prepared self-assembled monolayers with varying chemistries, i.e., SAMs-CH3, SAMs-NH2, SAMs-COOH and SAMs-OH, and characterized Fn adsorption and cell behaviors on them. Next, Monte Carlo method and all-atom molecular dynamics simulations were employed to reveal the orientation/conformation of Fn on surfaces. We found that SAMs-CH3 strongly adsorbed Fn via hydrophobic interactions, but show poor bioactivity as the low exposure of RGD/PHSRN motifs and the deformation of Fn. SAMs-NH2 and SAMs-COOH could adsorb Fn efficiently via vdW interactions, electrostatic interactions, hydrogen bonds and salt bridges. Fn exhibited excellent bioactivity for cell adhesion, proliferation and osteogenic differentiation as high exposure of bioactive motifs on SAMs-NH2, or as the activation of other inferior cell-binding motifs on SAMs-COOH. SAMs-OH showed poor Fn adsorption as the water film. However, the adsorbed Fn displayed non-negligible bioactivity due to high exposure of PHSRN motif and large degree of protein flexibility. We believe that the revealed mechanism presents great potential to rationally design Fn-activating biomaterials.