Cytoskeletal alterations in endothelial cells have been linked to nitric oxide generation and cell-cell interactions. Transforming growth factor (TGF)-β has been described to affect cytoskeletal rearrangement in numerous cell types; however, the underlying pathway is unclear. In the present study, we found that human umbilical vein endothelial cells (HUVEC) have marked cytoskeletal alterations with short-term TGF-β treatment resulting in filipodia formation and F-actin assembly. The cytoskeletal alterations were blocked by the novel TGF-β type I receptor/ALK5 kinase inhibitor (SB-505124) but not by the p38 kinase inhibitor (SB-203580). TGF-β also induced marked stimulation of reactive oxygen species (ROS) within 5 min of TGF-β exposure. TGF-β stimulation of ROS was mediated by the NAPDH oxidase homolog Nox4 as DPI, an inhibitor of NADPH oxidase, and dominant-negative Nox4 adenovirus blocked ROS production. Finally, inhibition of ROS with ROS scavengers or dominant-negative Nox4 blocked the TGF-β effect on cytoskeleton changes in endothelial cells. In conclusion, our studies show for the first time that TGF-β-induced ROS production in human endothelial cells is via Nox4 and that TGF-β alteration of cytoskeleton in HUVEC is mediated via a Nox4-dependent pathway.