The profound effects that nanoscale surface topography exerts on cell behavior are highly relevant to the development of advanced biomaterials and to advances in tissue engineering and regenerative medicine. Here, an asymmetric anodization procedure is used to produce n-type porous silicon (pSi) gradients with pore sizes ranging from tens to hundreds of nanometers in diameter and changes in the ridge nanoroughness from a few to tens nanometers. Rat mesenchymal stem cells (rMSCs) adhere poorly at the regions with small pore size but high ridge roughness. Cell adhesion is increased gradually towards the large pore size but low ridge roughness end of the pSi gradients. Surface topography influences cell differentiation, but not cell proliferation. Osteogenesis of rMSCs is enhanced by porous topography with a ridge roughness lower than 10 nm, while adipogenesis of rMSCs is enhanced on the entire pSi gradient compared with flat Si substrates. The results demonstrate that the gradient format allows in-depth screening of surface parameters that are important for the control of mammalian cell behavior, thereby advancing the development of new and improved biomaterials for orthopaedic and tissue engineering applications.