The ability to control the cellular response is of critical importance when designing advanced biomaterials for applications in tissue engineering and regenerative medicine. An important aspect of biointerfacial interactions is surface topography at the nanoscale and therefore this needs to be taken into consideration. Here, a pore size gradient in porous alumina (pAl) was fabricated with pore sizes ranging from 50 nm to 3 μm. The attachment behaviour and osteogenesis of human mesenchymal stem cells (hMSCs) was investigated along this topography gradient for up to 2 weeks. Generally, cell attachment density and spreading area decreased with increasing pore size. Pore wall width and solid surface fraction also played a key role in cell adhesion. After 2 weeks, osteogenesis of hMSCs was enhanced by porous topography with a pore size of 120-230 nm in diameter and 10 nm pore wall width, compared with other topographies of the gradient. The results demonstrate that the gradient format allows in-depth high-throughput screening of surface parameters that are important for the control of mammalian cell behaviour, thereby advancing the development of new and improved biomaterials for e.g. orthopaedic and tissue engineering applications.