Titanium (Ti) is one of the most used biomaterials in metals. However, Ti is typically artificial materials. Thus, it is necessary for improving the biocompatibility of Ti. Recently, coating of the titanium dioxides (TiO 2) film on Ti plate has been proposed to improve biocompatibility of Ti. We have developed coating method of the film on Ti plate with an aerosol beam. Periodic structures formation on biomaterials was also a useful method for improving the biocompatibility. Direction of cell spreading might be controlled along the grooves of periodic microstructures. In our previous study, periodic nanostructures were formed on the film by femtosecond laser irradiation at fundamental wave (775 nm). Period of the periodic nanostructures was about 230 nm. In 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. Then, influence of the period of the periodic nanostructures on cell spreading has not been investigated yet. The period might be changed by changing the laser wavelength. In this study, the periodic nanostructures were created on the film with femtosecond laser at 775nm and 388 nm, respectively. After cell test, cell spreading along the grooves of the periodic nanostructures was observed on 775 nm and 388nm laser irradiated areas. Distribution of direction of cell spreading on laser irradiated area was also examined. These results suggested that controlling the cell spreading on periodic nanostructures with period of 230 nm was better than that with period of 130 nm.