Titanium (Ti) is widely used in biomaterials because of its excellent anti-corrosion properties and high strength. However, Ti has no biological function, so its bioactivity must be improved. Coating a titanium dioxide (TiO2) film on a Ti plate surface has been shown to improve the biocompatibility of Ti plates. If periodic nanostructures were formed on the film surface, the direction of cell spreading might be controlled by the direction of the grooves. Controlling cell spreading on biomaterials would contribute to the creation of advanced biomaterials. In this paper, a TiO2 film was formed on a Ti plate with an aerosol beam composed of sub micron-sized TiO2 particles and helium gas. Periodic nanostructures, lying perpendicular to the laser electric field polarization vector, were formed on the film by scanning the femtosecond laser focusing spot. The period and height of the periodic nanostructures were about 230 nm and 150 nm, respectively. In a cell test, cell spreading was observed along the grooves of the periodic nanostructures; in contrast, cell spreading did not show a definite direction on TiO2 a film without periodic nanostructures. These results suggest that the direction of cell spreading on the film can be controlled by periodic nanostructure formation generated using a femtosecond laser. (C) 2013 Elsevier B. V. All rights reserved.