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Controlling neuronal cell adhesion, migration, and axonal outgrowth via contact interactions with biomaterials is a critical element for tissue engineering applications and for developing artificial neuronal interfaces. One promising approach relies on the exploitation of nanostructured surfaces. Here, the human neuroblastoma cell line SH‐SY5Y is interfaced with plastic nanogratings (NGs; anisotropic topographies composed by alternating lines of grooves and ridges with sub‐micrometer lateral dimension). The SH‐SY5Y cells' (SHs) contact guidance is investigated under proliferating conditions and upon differentiation after treatment with retinoic acid (RA) and brain‐derived neurotrophin factor (BDNF), and compared with mouse primary hippocampal neurons (HNs). Quantitative readouts are obtained by measuring changes in tubulin cytoskeleton organization and cell morphology induced by mechanotransduction …