Transforming growth factor‐β1 (TGF‐β1) is a cytokine critically involved in acute lung injury and endothelial cell (EC) barrier dysfunction. We have studied TGF‐β1‐mediated signaling pathways and examined a role of microtubule (MT) dynamics in TGF‐β1‐induced actin cytoskeletal remodeling and EC barrier dysfunction. TGF‐β1 (0.1–50 ng/ml) induced dose‐dependent decrease in transendothelial electrical resistance (TER) in bovine pulmonary ECs, which was linked to increased actin stress fiber formation, myosin light chain (MLC) phosphorylation, EC retraction, and gap formation. Inhibitor of TGF‐β1 receptor kinase RI (5 μM) abolished TGF‐β1‐induced TER decline, whereas inhibitor of caspase‐3 zVAD (10 μM) was without effect. TGF‐β1‐induced EC barrier dysfunction was linked to partial dissolution of peripheral MT meshwork and decreased levels of stable (acetylated) MT pool, whereas MT stabilization by taxol (5 μM) attenuated TGF‐β1‐induced barrier dysfunction and actin remodeling. TGF‐β1 induced sustained activation of small GTPase Rho and its effector Rho‐kinase; phosphorylation of myosin binding subunit of myosin specific phosphatase; MLC phosphorylation; EC contraction; and gap formation, which was abolished by inhibition of Rho and Rho‐kinase, and by MT stabilization with taxol. Finally, elevation of intracellular cAMP induced by forskolin (50 μM) attenuated TGF‐β1‐induced barrier dysfunction, MLC phosphorylation, and protected the MT peripheral network. These results suggest a novel role for MT dynamics in the TGF‐β1‐mediated Rho regulation, EC barrier dysfunction, and actin remodeling. © 2005 Wiley‐Liss, Inc.