Mammalian cells respond to their substrates by complex changes in gene expression profiles, morphology, proliferation and migration. We report that substrate nanotopography alters morpohology and proliferation of human embryonic stem cells (hESCs). Fibronectin-coated poly(di-methyl siloxane) substrates with line-grating (600nm ridges with 600nm spacing and 600±150nm feature height) induced hESC alignment and elongation, mediated the organization of cytoskeletal components including actin, vimentin, and α-tubulin, and reduced proliferation. Spatial polarization of gamma-tubulin complexes was also observed in response to nanotopography. Furthermore, the addition of actin disrupting agents attenuated the alignment and proliferative effects of nanotopography. These findings further demonstrate the importance of interplay between cytoskeleton and substrate interactions as a key modulator of morphological and proliferative cellular response in hESCs on nanotopography.