TiO2 anodized nanotubelayers are potentially useful for orthopedic and dental implants because they promotes bone formation. Peptide sequences, such as lysine-arginine-serine-arginine (KRSR), are often used to modify biomaterial surfaces for the selective adhesion of bone cells. The objective of this study was to functionalize TiO2 nanotube layers with KRSR to examine the responses of mouse preosteoblasts (MC3T3-E1) to this new material in vitro. SEM, AFM, XPS were used to characterize the materials. Immunofluorescence staining, SEM, ALP, RT-PCR, Wb analysis were used to detect the preosteoblast adhesion, spreading and osteogenic differentiation. KRSR peptides could be immobilized on the TiO2 nanotube layers by silane coupling. Immobilized KRSR increased preosteoblast adhesion and spreading on TiO2 nanotube layers. Moreover, osteogenic differentiation increased on the KRSR-modified TiO2 nanotube layers. KRSR-modified TiO2 nanotube layers have satisfactory biological properties and should be further investigated as medical implant materials.