Peritoneal dysfunction is a major factor leading to treatment failure of peritoneal dialysis (PD). However, the precise mechanism of the peritoneal diffusion changes related to PD remains to be elucidated. To this end, we have established a novel peritoneal diffusion model in vitro, which consists of a three-dimensional culture system using a collagen vitrigel membrane chamber and a fluid-stream generation system. This artificial peritoneal model revealed that high-glucose culture medium and fluid flow stress promoted the epithelial-mesenchymal transition (EMT) process of mesothelial cells, and that endothelial cells inhibited this mesothelial EMT process. Mesothelial cells in the EMT state showed high expression of connective tissue growth factor and low expression of bone morphogenic protein-7, while non-EMT mesothelial cells showed the opposite expression pattern of these two proteins. In addition, these protein expressions were dependent on the presence of endothelial cells in the model. Our model revealed that the endothelial slit function was predominantly dependent on the covering surface area, while the mesothelial layer possessed a specific barrier function against for small solutes independently of the surface area. Notably, a synergic barrier effect of mesothelial cells and endothelial cells was present with low glucose pretreatment, but high glucose pretreatment abolished this synergic effect. These findings suggest that the mesothelial slit function is not only regulated by the high-glucose-induced EMT process, but is also affected by an endothelial paracrine effect. This peritoneal diffusion model could be a promising tool for the development of PD.