We cultured human mesenchymal stem cells (hMSCs) on TiO2 nanotubes with diameters of 30–100 nm to assess the size-effect of TiO2 nanotubes on the behavior and osteogenic functionality of hMSCs. Most studies of the expression of genes encoding alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), and integrin-β (INT-B), after 1 week of incubation, supported the results of cell viability and MTT assays at 48 hrs of plating. However, after 2 weeks of incubation, expression of ALP, OPN, INT-B, and protein kinase R-like ER kinase (PERK) genes were significantly higher in cells cultured on 70 nm TiO2 nanotubes than that in cells cultured on other TiO2 nanotubes and Ti. This biphasic osteogenic characteristic of hMSCs is supposed to relating to the nature of the hMSCs adhering to the substrate at the beginning of incubation, and the nanostructural stimulation caused by the topography of TiO2 nanotubes at a later stage of incubation. The discovery of these biphasic characteristics of hMSCs cultured on different-sized TiO2 nanotubes may contribute to resolving the discrepant results relating to the size-effect of TiO2 nanotubes on the adhesion, proliferation, and functionality of cells.