The hydroxyapatite (HAp) formation ability after soaking in a simulated body fluid, MC3T3-E1 cell differentiation and related surface potentials of rutile-type TiO 2 surfaces formed on Ti are controlled by varying the Ti heat treatment conditions in a N 2 atmosphere containing a trace amount of O 2. The zeta potentials of the samples heated at 873 and 973 K for 1 h are large negative and positive values, respectively. Scanning transmission electron microscopy, electron energy loss spectroscopy, and calculations of defect formation energies reveal that nitrogen atoms incorporated into TiO 2 during surface formation produce the charged defects (NO) O −1 and (N 2) O +2 for the scales formed in 1 h at 873 and 973 K, respectively. HAp formation and MC3T3-E1 cell differentiation is more pronounced on charged nitrogen-doped TiO 2 surfaces compared to on an untreated, neutral Ti surface. An adhesive protein such as Fn adsorbs equally on charged and untreated Ti surfaces in culture medium. However, the adsorption of Ca and P was only detected on charged nitrogen-doped TiO 2 surfaces. The enhanced adsorption of inorganic ions and Fn is probably responsible for promoting initial stage of osteoblast differentiation. The present findings lead to physical models of surface charge distributions that elucidate the relationship between nitrogen-related defects, charged surfaces and cell differentiation mechanisms.