There are few methodologies that allow manipulating a biomaterial surface at nanometer scale, which controllably influence different cellular functions. In this study, virus nanoparticles with different structural features are selected to prepare 2D substrates with defined nanoscale topographies and the cellular responses are investigated. It is demonstrated that the viral nanoparticle based substrates could accelerate and enhance osteogenesis of bone derived mesenchymal stem cells as indicated by the upregulation of osteogenic markers, including bone morphogenetic protein‐2, osteocalcin, and osteopontin, at both gene and protein expression levels. Moreover, alkaline phosphatase activity and calcium mineralization, both indicators for a successful bone formation, are also increased in cells grown on these nanoscale possessed substrates. These discoveries and developments present a new paradigm for nanoscale engineering of a biomaterial surface.