The lack of biological activity of pure titanium as a dental implant has attracted increasing attention. This condition can be improved by preparing a layer of nano-hydroxyapatite (nano-HA) coating that is stable and does not easily fall off, which however, remains a challenge. Here, we report the preparation of nano-HA crystals on calcified titanium oxide (TiO2) nanotube arrays by means of anodization, heat treatment, calcification in vacuum and hydrothermal phosphorylation, achieving the chemical bonding between the nano-HA coating and TiO2 substrate. The mechanism of coating formation was also investigated. The results confirmed that annealing at 450 degrees C could convert the amorphous nanotubes to the anatase structure. Calcium titanate (CaTiO3) was formed on the surface of nanotubes by heat treatment after calcification, which could provide nucleation sites for the formation of nano-HA. Needle-like nano-HA crystals were subsequently formed on the surface of the TiO2 nanotube arrays. The scratch test showed that the adhesion strength between the formed nano-HA coating and the TiO2 nanotube arrays was higher than 29.17 +/- 1.07 N. In vitro cell proliferation and adhesion experiments showed that the nano-HA coating had good biocompatibility. Alkaline phosphatase (ALP) activity and osteocalcin (OCN) expressions highlighted the higher osteoconductivity of the nano-HA coating on the surface of TiO2 nanotube arrays compared to the pure Ti.