The effect of anodic oxide films produced by β-glycerophosphate (β-GP) and calcium acetate (CA) anodizing on osteoblast-like cell attachment and spreading were evaluated in this study. Anodic oxide films were produced in different conditions: Group 1, 0.02 M β-GP and 0.2 M CA; Group 2, 0.03 M β-GP and 0.2 M CA; Group 3, 0.03 M β-GP and 0.2 M CA. Anodic oxide surface was significantly rougher in comparison to the control untreated titanium surfaces, and the surface roughness and composition of phosphate and oxide increased as the concentration of β-GP was increased. There was no significant difference in the cell viability when cells were cultured on the control or anodized surface using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Scanning electron micrographs revealed more spread cells on the anodized surface than on the smooth control surface. In conclusion, we suggested that the positive effects of anodized surfaces produced by β-GP and CA on spreading of osteoblast-like cells may be the result of the difference of surface roughness and amount of Ca and P in the oxide layer.