Titanium (Ti) is an important biomaterial. We have used femtosecond laser irradiation to form periodic nanostructures on Ti plate for control of the cell spreading. In this study, periodic nanostructures with periodicities of 200, 300 and 600 nm were formed on a Ti plates using a femtosecond laser with wavelengths of 258, 388 and 775 nm, respectively. Cell spreading on the Ti plate for periodic nanostructures with periodicity of 200 nm lacked a definite direction, whereas cell spreadings on the Ti plate for periodic nanostructures with periodicities of 300 and 600 nm occurred along the grooves.