The pore diameter of uniformly structured nanotubes can significantly change the behaviour of cells. Recent studies demonstrated that the activation of integrins is affected not by only the surface chemistry between the cell-material interfaces, but also by the features of surface nanotopography, including nanotube diameter. While research has been carried out in this area, there has yet to be a single systemic study to date that succinctly compares the response of both human stem cells and osteoblasts to a range of TiO2 nanotube pore diameters using controlled experiments in a single laboratory. In this paper, we investigate the influence of surface nanotopography on cellular behaviour and osseointegrative properties through a systemic study involving human mesenchymal stem cells (hMSCs) and human osteoblasts (HOBs) on TiO2 nanotubes of 20 nm, 50 nm and 100 nm pore diameters using in-vitro assessments. This detailed study demonstrates the interrelationship between cellular behaviour and nanotopography, revealing that a 20 nm nanotube pore diameter is preferred by hMSCs for the induction of osteogenic differentiation, while 50 nm nanotubular structures are favourable by HOBs for osteoblastic maturation.