Surface roughness affects various cell activities, including osteoblast motility, which may have an effect on bone regeneration. Defective cell signaling, which is associated with the slow motility of osteoblasts on a substrate with rough topology at nanometer dimensions (Ra = 123.8 ± 29.1 nm), was studied. Osteoblasts grown on the rough surface at nanometer dimensions showed the high activities of small GTPase RhoA and Rho-associated kinase (ROCK) on the rough surface at nanometer dimensions and downregulated Rac1 activity compared to the smooth surface. The inhibition of ROCK in the cells with Y-27632, a specific ROCK inhibitor, reversed the low-cell motility. In addition, the transfection of constitutively active Rac1 reversed the low-cell motility. However, Rac1 inhibition abolished the reversal of low-cell motility induced by ROCK inhibition. These results indicate that upregulated RhoA/ROCK activity suppresses Rac1 activity to decrease the motility of osteoblasts on a rough surface at nanometer dimensions, and the low motility of osteoblasts on a rough surface at nanometer dimensions can be reversed by ROCK inhibition. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.