Transforming growth factor-b (TGF-b) and downstream Smad signaling pathways have been found to be the most important pathways involved in tissue fibrosis via induction of the profibrotic genes connective tissue growth factor (CTGF) and tissue inhibitors of matrix metalloproteinase-1 (TIMP-1). Cyclosporin A (CsA) is one of the most efficient immunosuppressive drugs that has been shown to induce a rapid activation of TGF-b/Smad signaling pathway. The present work demonstrates that CsA similar to TGF-b causes a rapid phosphorylation of Smad-2 in A549 lung epithelial cells which is abrogated by the addition of neutralizing TGF-b-antibody. By using the TGF-b receptor I kinase inhibitor, activin receptor-like kinase-5 (ALK-5) a critical involvement of TGF-b receptor in CsA-triggered Smad signaling was depicted. Furthermore, TGF-b release and Smad-2 phosphorylation induced by CsA were highly reduced in the presence of the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC), indicating that ROS is required for TGF-b release and Smad-2 phosphorylation induced by CsA. Moreover, the present work demonstrates that CsA via ROS generation and activation of TGF-b/Smad signaling cascade can cause an increase in the expression of the profibrotic genes CTGF and TIMP-1 in A549 lung epithelial cells. Collectively, these data demonstrate that CsA causes a rapid activation of the fibrogenic Smad signaling cascade in A549 lung epithelial cells via generation of ROS and subsequent activation of latent TGFβ that is sufficient to elicit fibrogenic cell responses as indicated by increasing the expression of the fibrogenic genes CTGF and TIMP-1 that play an important role in tissue fibrosis.