Mesoporous hydroxyapatite (meso-HA) thin films were fabricated by a sol-gel method using cetyltrimethyl ammonium bromide as the template. The phase, surface morphology, and mesoporous structure of the meso-HA films were characterized by X-ray diffraction, scanning electron microscopy, and transmission electron microscopy. The uniform thin films consisted of meso-HA spherical particles with different pore size (2.0 or 3.1 nm) were prepared on the glass substrate at different pH value (pH 3.0 or 7.0). The introduction of mesopores would enhance the surface area of HA. Water contact angle was also measured on the non-mesoporous and meso-HA thin films, revealing the promotion of surface wettability in the mesoporous ones. In vitro cytocompatibility of HA films were evaluated by cell adhesion and proliferation tests using MC3T3-E1 cells. After 3 days of culture on the samples, the cells spread in an elongated shape and were well adhered to the surface of the meso-HA films. Moreover, the cells proliferation on the meso-HA films was higher than that on the non-mesoporous films. There are significant differences in the cell density between the control group and the meso-HA films with the pores sized in 2.0 nm after being cultured for 2 and 3 days (P < 0.05). The results suggested that the presence of mesopores could influence the surface and biological properties of HA films, and the mesoporous structure would enhance the cell response of HA.