Aligned or random nanofibrous meshes of multiwalled carbon nanotubes/polyurethane composite (MWCNT/PU) were fabricated by electrospinning and characterized by scanning electron microscopy (SEM). The regulatory effects of nanofibrous structure and MWCNT on the growth and anticoagulant function of human umbilical vein endothelial cells (HUVECs) were investigated by examining proliferation, type IV collagen secretion, tissue factor and plasminogen activator inhibitor-1 (PAI-1) release, and cytoskeleton arrangement, as well as via pull-down analysis. We show that aligned nanofibrous structure and MWCNT can function as extracellular signals to stimulate cell growth, proliferation, and extracellular collagen secretion, in addition to preserving anticoagulant function. The nanofibrous structures played important roles in the activation of Rac and Cdc42, while CNT regulated the activation of Rho. These two features synergistically activated Rho GTPases that transmitted cell-substrate signals to the cytoplasm. These signals were then relayed to the nucleus by the MAP kinase pathway to direct cytoskeletal arrangement and cell orientation. (C) 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 95A: 312-320, 2010.