Osteopontin (OPN) is an important protein for mediating cell behaviour on biomaterials. However, the interactions between the chemical groups on the biomaterial surface and OPN still need to be further clarified, which has restricted the application of OPN in biomaterial functionalization. In the present study, we developed different self-assembled monolayers (SAMs) with specific chemical groups, including SAMs-OH, SAMs-OEG, SAMs-COOH, SAMs-NH₂, and SAMs-PO₃H₂, to study the behavior of OPN on these SAMs. The results showed that SAMs-NH₂ could strongly adsorb OPN, and the amount of protein was highest on this material. Meanwhile, the lowest amount of OPN was present on SAMs-OEG. Interestingly, the unit-mass trend of bound OPN monoclonal antibodies (mAbs) on the SAMs was opposite to the OPN adsorption trend: lowest on SAMs-NH₂ but highest on SAMs-OEG. In vitro cell assay results showed that mouse bone marrow mesenchymal stem cells (mBMSCs) on SAMs-COOH, SAMs-NH₂, and SAMs-PO₃H₂ with pre-adsorbed OPN showed promoted behaviour, in terms of spreading, viability, and the expression levels of α_v and β₃ genes, compared with the other two SAMs, demonstrating the higher bioactivity of the adsorbed OPN. We believe that our findings will have great potential for developing OPN-activated biomaterials.