The extracellular matrix exhibits several nanostructures such as fibers, filaments, nanopores and ridges that can be mimicked by topographical and 3D substrates for cell and tissue cultures for an environment closer to in vivo conditions. This review summarizes and discusses a growing number of reports employing nanoimprint lithography to obtain such scaffolds. The different nanoimprint lithography methods as well as their advantages and disadvantages are described and special attention is paid to cell culture applications. We discuss the impact of materials, nanotopography, size, geometry, fabrication method, and cell type on growth guidance and differentiation. We present examples of cell guidance, inhibition of cell growth, cell pinning and engineering of 3D cell sheets or spheroids. As current applications are limited and not systematically compared for various cell types, this review only suggests promising substrates for particular applications. Future possible directions are also proposed in which this field may proceed.