Endothelial-mesenchymal transition (EndMT) plays important roles in various physiological and pathological processes. While signals mediated by transforming growth factor (TGF)-β have been implicated in EndMT, the molecular mechanisms underlying it remain to be fully elucidated. Here, we examined the effects of TGF-β signals on the EndMT of mouse pancreatic microvascular endothelial cells (MS-1). By addition of TGF-β2, MS-1 cells underwent mesenchymal transition characterized by re-organization of actin stress fibre and increased expression of various mesenchymal markers such as α-smooth muscle actin (α-SMA) through activation of Rho signals. Whereas activation of Rho signals via TGF-β-induced non-Smad signals has been implicated in epithelial-mesenchymal transition (EMT), we found that Arhgef5, a guanine nucleotide exchange factor, is induced by Smad signals and contributes to the TGF-β2-induced α-SMA expression in MS-1 cells. We also found that TGF-β2 induces the expression of myocardin-related transcription factor-A (MRTF-A) in a Smad-dependent fashion and its nuclear accumulation in MS-1 cells and that MRTF-A is required and sufficient for TGF-β2-induced α-SMA expression. These results indicate that activation of Smad signals by TGF-β2 have dual effects on the activation of Rho signals and MRTF-A leading to the mesenchymal transition of MS-1 endothelial cells.