In this study, we report an efficient and cost-effective method of fabricating polystyrene (PS) nano-featured substrates containing nanopore (NPo) and nanopillar (NPi) arrays based on hot embossing using nickel nano-stamps. We investigate the behavior of adipose-derived stem cells (ASCs), including adhesion, morphology, proliferation and differentiation, on the replicated PS surfaces. Compared to a flat substrate, NPo- and NPi-featured substrates do not alter the morphology of stem cells. However, both NPo- and NPi-featured substrates induce different integrin expression and lower formation of focal adhesion complexes. In addition, ASCs on the NPo-featured substrate exhibit greater adipogenic differentiation, while the NPi-featured substrate induces higher osteogenic differentiation.