Titiania nanotubes have large potential in medical implant applications but their tissue compatibility is still controversial. Since the sterilization methods may impact the biocompatibility of titania nanotubes and be the source of the controversy, we investigate the influence of three commonly used sterilization methods, autoclaving, ultraviolent irradiation and ethanol immersion, on the cytocompatibility of titania nanotubes. Two titania nanostructures, namely nanonets with an average pore diameter of 25 nm and nanotubes with an average diameter of 80 nm, are used in this study. The results show that the sterilization methods significantly affect the cytocompatibility of these titania surfaces. UV and ethanol sterilization give rise to a higher surface free energy inducing higher initial cell adhesion and proliferation compared to autoclaving, whereas UV irradiation produces the best cell functions including adhesion, proliferation, as well as differentiation represented by related gene expressions. The cytocompatibility results obtained from the nanoscale surfaces are compared to those acquired from the polished surface demonstrating the significant effects. Our results suggest that the sterilization process plays an important role in the observed cytocompatibility of titania nanotubes and may be the reason for the controversial results so far. UV sterilization is found to be the best method from the viewpoint of surface contamination elimination. © 2009 Elsevier Ltd. All rights reserved.