Titanium (Ti) is widely used in the field of hard tissue implant materials due to its desirable mechanical properties and biocompatibility, but its osteogenic activity is relatively poor, hindering its clinical applications. In this work, Na-Ti-O nanostructured film was prepared on Ti surface by anodization in 2 M NaOH electrolyte at different applied voltages. The results show that uniform film can be formed when the voltage is between 15 and 25 V. Compared with untreated Ti, the anodized samples can promote osteoblast spreading, proliferation, and osteogenic differentiation. These results indicate Na-Ti-O nanostructured film is promising to functionalize Ti-based materials for dental and orthopedic applications. •Na-Ti-O nanostructured film was anodically grown on titanium surface in NaOH electrolyte.•The film could be grown at applied voltage of 15–25 V with the NaOH concentration of 2 M.•The film could promote osteoblast spreading, proliferation, and osteogenic differentiation.