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Live cells are exquisitely sensitive to both the sub- stratum rigidity and texture. To explore cell responses to both these types of inputs in a precisely controlled fashion, we analyzed the responses of Chinese hamster ovary (CHO) cells to nanotopographically defined substrata of different rigidities, ranging from 1.8 MPa to 1.1 GPa. Parallel arrays of nanogrooves (800-nm width, 800-nm space, and 800-nm depth) on polyurethane (PU)-based material surfaces were fabricated by UV-assisted capillary force lithography (CFL) over an area of 5 mm × 3 mm. We observed dramatic morphological responses of CHO cells, evident in their elongation and polarization along the nanogrooves direction. The cells were progressively more spread and elongated as the sub- stratum rigidity increased, in an integrin β1 dependent manner. However, the degree of orientation was independent of substratum rigidity, suggesting that the cell shape is primarily determined by the topographical cues.