Peritoneal fibrosis, a major complication of peritoneal dialysis, limits the effectiveness of peritoneal dialysis as a treatment of end-stage renal disease. Preventing this complication by identifying targets for therapy has recently received much attention. In the present study, we showed that Notch signaling was highly activated in rats in peritoneal dialysis fluid-induced fibrotic peritoneum, as indicated by increased expression of Jagged-1, Notch-1, and HES-1. Blocking Notch signaling activation by intraperitoneal injection of a gamma-secretase inhibitor, DAPT, significantly attenuated peritoneal fibrosis as indicated by the decreased expression of alpha-smooth muscle actin, collagen I, and vascular endothelial growth factor as well as increased expression of E-cadherin. Moreover, compared with control rats, DAPT-treated rats had a thinner peritoneum with less extracellular matrix accumulation, a lower mass transfer of glucose, and a higher ultrafiltration rate. In addition, transforming growth factor (TGF)-beta1 induced Notch signaling activation in primary rat peritoneal mesothelial cells. DAPT blocked this TGF-beta1-induced Notch signaling activation and therefore significantly inhibited TGF-beta1-induced expression of alpha-smooth muscle actin, collagen I, and vascular endothelial growth factor. Thus, a gamma-secretase inhibitor that interferes with Notch signaling prevents biochemical, histological, and functional consequences of peritoneal fibrosis through inhibiting epithelial to mesenchymal transition of rat peritoneal mesothelial cells. These results support the use of gamma-secretase inhibitors as a novel therapeutic approach for peritoneal fibrosis.