Nanoscale cell-substratum interactions are of significant interest in various biomedical applications. We investigated human foetal osteoblastic cell response to randomly distributed nanoisland topography with varying heights (11, 38 and 85 nm) produced by a polystyrene (PS)/polybromostyrene polymer-demixing technique. Cells displayed island-conforming lamellipodia spreading, and filopodia projections appeared to play a role in sensing the nanotopography. Cells cultured on 11 nm high islands displayed significantly enhanced cell spreading and larger cell dimensions than cells on larger nanoislands or flat PS control, on which cells often displayed a stellate shape. Development of signal transmitting structures such as focal adhesive vinculin protein and cytoskeletal actin stress fibres was more pronounced, as was their colocalization, in cells cultured on smaller nanoisland surfaces. Cell adhesion and proliferation were greater with decreasing island height. Alkaline phosphatase (AP) activity, an early stage marker of bone cell differentiation, also exhibited nanotopography dependence, i.e. higher AP activity on 11 nm islands compared with that on larger islands or flat PS. Therefore, randomly distributed island topography with varying nanoscale heights not only affect adhesion-related cell behaviour but also bone cell phenotype. Our results suggest that modulation of nanoscale topography may be exploited to control cell function at cell-biomaterial interfaces.