High-aspect-ratio nanostructures have emerged as versatile platforms for intracellular sensing and biomolecule delivery. Here, we present a microfabrication approach in which a combination of reactive ion etching protocols was used to produce high-aspect-ratio, nondegradable silicon nanoneedle arrays with tip diameters that can be finely tuned between 20 and 700 nm. We used these arrays to guide the long-term culture of human mesenchymal stem cells (hMSCs). Notably, we used the nanoneedle tip diameter to control the morphology, nuclear size and F-actin alignment of interfaced hMSCs, and to regulate the expression of nuclear lamina genes, Yes-associated protein (YAP) target genes and focal adhesion genes. These topography-driven changes were attributed to signaling by Rho-family GTPase pathways, differences in the effective stiffness of the nanoneedle arrays and the degree of nuclear membrane impingement, with the latter clearly visualized using focused-ion beam scanning electron microscopy (FIB-SEM). Our approach to design high-aspect-ratio nanostructures will be broadly applicable to design biomaterials and biomedical devices used for long-term cell stimulation and monitoring.