Periodic nanostructures formation on Titanium dioxide (TiO2) film by scanning of femtosecond laser beam spot at fundamental and second harmonic wave is reported. Titanium (Ti) is one of the most widely used for biomaterials, because of its excellent anti-corrosion and high mechanical properties. However, Ti implant is typically artificial materials and has no biofunction. Hence, it is necessary for improving the bioactivity of Ti. Recently, coating of TiO2 film on Ti plate surface is useful methods to improve biocompatibility of Ti plate. Then, if periodic nanostructures were formed on the film surface, cell spreading might be controlled at one direction. We propose periodic nanostructures formation on TiO2 film by femtosecond laser irradiation. Cell spread could be controlled along the grooves of periodic nanostructures. In the experiments, the film was formed on Ti plate with an aerosol beam. A commercial femtosecond Ti : sapphire laser system was employed in our experiments. Periodic nanostructures, lying perpendicular to the laser electric field polarization vector, were formed on the film at fundamental and second harmonic wave. Periodic nanostructures were also produced on Ti plate with femtosecond laser. The period of periodic nanostructures on the film was much shorter than that on Ti plate. By cell test, there was a region of cell spreading along the grooves of periodic nanostructures on the film formed with femtosecond laser at fundamental wave. On bare film surface, cell spreading was observed at all direction. These results suggest that direction of cell spread could be controlled by periodic nanostructures formation on the film.