The surface properties of biomaterials play a vital role in cell morphology and behaviors such as cell adhesion, migration, proliferation and differentiation. Three different crystal phases of titania film (rutile, anatase and amorphous titania) with similar roughness were successfully synthesized by DC reactive magnetron sputtering. The surface roughness of each film was about 8-10 nm. Primary rat osteoblasts were used to observe changes in morphology and to evaluate cell behavior at the film surface. The number of the osteoblasts on anatase film was significantly higher than rutile and amorphous films after 36 and 72 h incubation. More importantly, synthesis of alkaline phosphatase was significantly greater by osteoblasts cultured on anatase film than on rutile and amorphous films after 7 and 14 days. In addition, the cells grown on the anatase phase film had the largest spreading area; the actin filaments in cells with regular directions were well defined and fully spreaded. The results indicate that the anatase phase of titania with nanoscale topography yield the best biological effects for cell adhesion, spreading, proliferation and differentiation. There are strong therapeutic prospects for this biomaterial film for osteoblast proliferation, with possible applications for orthopedic and dental implant.