Fibronectin (FN) containing a heparin-binding domain (HBD) and an Arg-Gly-Asp (RGD) domain can promote cell adhesion and proliferation compared to FN that contained only RGD. Here, we have engineered recombinant human osteocalcin (rhOC) with FN type III9-14 (rhOC-FNIII9-14) containing RGD and HBD to promote the cellular activity of MC3T3-E1 cells, including adhesion, proliferation, and differentiation. RhOC-FNIII9-14 significantly increased cell adhesion and proliferation of MC3T3-E1 cells compared to rhOC-FNIII9-10 (P < 0.05). Moreover, rhOC-FNIII9-14 showed osteogenic differentiation of MC3T3-E1 cells in mineralization activity and osteogenic gene expression.