Objective To develop a reliable method for primary culture of normal human peritoneal mesothelial cells. Methods Human peritoneal mesothelial cells were dissociated by a mixture of pancreatin and ethylene diamine tetraacetic acid with a magnetic puddler. Inverted phase contrast microscope was used to observe the morphological structures of cells, approximate process of growth. Calretinin was used to identify the mesothelial cells. Results On the 4th d of culture, mesothelial cells adhered to the culture dish. After day 14, mesothelial cells confluenced gradually and grew well like the slabstone. Calretinin was positively expressed by mesothelial cells after 5 d of cultivation. The mesothelial cell population of subculture was less than that of the primary culture. Conclusion A reliable method for primary culture of normal human peritoneal mesothelial cells has been successfully developed, by which sufficient amount of highly purified normal human peritoneal mesothelial cells can be obtained.