The interaction between stem cells and biomaterials with nanoscale topography represents a main route in the roadmap for tissue engineering-based strategies. In this study, we explored the interface between human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and hydrogenated amorphous carbon (a-C:H) film designed with uniform, groove, or grid nanopatterns. In either case, hBM-MSCs preserved growth rate and multi-differentiation properties, suggesting that the films were biocompatible and suitable for stem cell culture. hBM-MSCs responded to different nanopattern designs with specific changes of microtubule organization. In particular, the grid pattern induced a square-localized distribution of alpha-tubulin/actin fibers, whereas the groove pattern exerted a more dynamic effect, associated with microtubule alignment and elongation.