Peritoneal dissemination is one of the leading causes of death in gastric cancer patients. The interaction between carcinoma cells and the peritoneal lining may play a key role in tumor peritoneal dissemination. Human peritoneal mesothelial cells are a monolayer of squamous epithelial cells covering the peritoneal cavity and forming serosal membranes. The precise role of mesothelial cells in the peritoneal dissemination of gastric cancer remains to be identified. Expression of TGF-ß1, a cytokine known for its capacity to induce proliferative and transformative changes in cells, has been correlated with peritoneal metastasis and TNM stages of gastric cancer. High levels of TGF-ß1 in the subperitoneal milieu may play a key role in the transition of normal mesothelial cells to myofibroblasts. Here, we demonstrate that mesothelial cells activated by TGF-ß1 undergo epithelial-mesenchymal transition (EMT) and that the transition of mesothelial cells to myofibroblasts is dependent on Smad2 signaling. EMT of mesothelial cells was marked by up-regulation of α-smooth muscle actin and vimentin expression. Cytokeratin and E-cadherin expression decreased over time in transformed mesothelial cells. Knockdown of Smad2 gene by siRNA silencing significantly suppressed the transition of mesothelial cells to myofibroblasts. We conclude that when exposed to TGF-ß1 mesothelial cells undergo EMT which involves Smad2 signaling. Furthermore, mesothelial cells may be the possible source of myofibroblasts in peritoneal fibrosis and provide a favorable environment for the dissemination of gastric cancer.