Abstract Cells in their native microenvironment interact with three-dimensional (3D) nanofeatures. Despite many reports on the effects of substrate nanotopography on cells, the independent effect of 3D parameters has not been investigated. Recent advances in nanofabrication for precise control of nanostructure pattern, periodicity, shape, and height enabled this systematic study of cell interactions with 3D nanotopographies. Two distinct nanopatterns (posts and grates) with varying three-dimensionalities (50–600 nm in nanostructure height) were created, while maintaining the pattern periodicity (230 nm in pitch) and tip shape (needle- or blade-like sharp tips). Human foreskin fibroblasts exhibited significantly smaller cell size and lower proliferation on needle-like nanoposts, and enhanced elongation with alignment on blade-like nanogrates. These phenomena became more pronounced as the nanotopographical three-dimensionality (structural height) increased. The nanopost and nanograte architectures provided the distinct contact guidance for both filopodia extension and the formation of adhesion molecules complex, which was believed to lead to the unique cell behaviors observed.