MC3T3-E1 cell differentiation is more pronounced after 14 days incubation on charged nitrogen-doped TiO₂ surfaces compared to on an untreated, neutral Ti surface. The protein fibronectin (Fn) was detected by an immunogold-labeling technique and Ca and P were detected by time-of-flight secondary ion mass spectrometry. Both techniques revealed that an adhesive protein such as Fn adsorbs equally on negatively-charged, positively-charged, and untreated Ti surfaces in culture medium. However, the adsorption of Ca and P was only detected on charged nitrogen-doped TiO₂ surfaces. The enhanced adsorption of inorganic ions and Fn is probably responsible for promoting initial stage of osteoblast differentiation. The conformation of adsorbed Fn was observed by high-speed atomic force microscopy and found to be in the side-on orientation on the positively-charged surface. This finding may help elucidate the relation between Fn conformation and cell activity on surface potential-controlled nitrogen-doped TiO₂ surfaces in future.