Effects of nanometer-scale surface roughness of titanium oxide layer on the adhesion and proliferation of osteoblast-like cell were examined. The nano-structured titanium oxide layer was prepared on titanium substrates by chemical treatment and heat-treatment. The anatase or rutile phase of titanium oxide was formed on the titanium substrates. The surface roughness of titanium oxide layer depended on the chemical treatment time or the heat-treatment time. The average roughness (Sa) was controlled in the range from 10 to 70 nm. The cell metabolic activity increased on the substrate with limited surface roughness. It is found that cell proliferation is affected by nanometer-scale surface roughness of titanium oxide layer.