Zinc oxide (ZnO) has attracted a great deal of interest due to its electronic and optical properties, and has potential applications in biomedical field, while few studies have been conducted to investigate its biocompatibility. In this study, we prepared nanostructured ZnO on acid-etched pure Ti surface through a moderate hydrothermal treatment in ammonia solution (pH = 12.6). By changing Zn precursor concentration and hydrothermal duration, it was feasible to synthesis flake-like ZnO nanostructures with variable spacings. The adhesion, proliferation and alkaline phosphatase (ALP) activity of SaOS-2 cells were enhanced on the ZnO nanostructures when the Zn precursor concentration was 0.02 M and the hydrothermal duration was 4 h, compared to those on the acid-etched pure Ti without ZnO nanostructures. Narrower spacings (< 70 nm) between ZnO nanostructures are considered to contribute to this enhancement. Together, the results indicate that the superposition of ZnO nanostructures on titanium surfaces may be beneficial for the enhanced biological performance.