Titanium (Ti) is one of the most widely used for biomaterials, because of its excellent anti-corrosion and high mechanical properties. In addion to these properies, the bioactivity of Ti is required. Recently, coating of the titanium dioxide (TiO2) film on Ti plate surface is useful methods to obtain biocompatibility of Ti plate. If periodic nanostructures were formed on the film surface, direction of cell spreading might be controlled due to grooves direction. Then, femtosecond laser is one of the useful tools of periodic nanostructures formation. Peiriod of periodic nanostructures might be varied by changing the laser wavelength. In the experiments, the film was formed on Ti plate with an aerosol beam which was composed of submicron size TiO2 particles and helium gas. The film was irradiated with the femtosecond laser. Laser wavelengths of the laser was at 1044, 775 and 388 nm, respectively. Periodic nanostructures, lying perpendicular to the laser electric field polarization vector, were formed on the film by femtosecond laser irradiation at 1044, 775 and 388 nm, respectively. The period of the periodic nanostructures on the film produced by femtosecond laser irradiation at 1044, 775 and 388 nm was about 350, 230 and 130 nm, respectively. In the cell test, cell spreading along the grooves of the periodic nanostructures was observed although it was not done for the film without the periodic nanostructures. These results suggested that direction of cell spreading could be controlled by the periodic nanostructures formation.