The aim of this study was to investigate the secretion, structure and function of peritrophic membrane (PM) in digestive tract of Eriocheir sinensis. Histology and electron microscopy techniques were employed to observe the intestinal and PM morphology of E. sinensis. Chitinase dissolution test and SDS-PAGE gel electrophoresis were used to research the composition of the PM and then the function of PM protein was indentified by LC-ESI-MS/MS. Microscopic examination showed the surfaces of gastrointestinal tract of E. sinensis were all covered with PM. Compartmentalization of PM was observed in intestine except midgut. When PM was detached from the epithelial cells, the columnar epithelial cells were becoming irregular round with the nucleus disappeared. A large number of mitochondria and secretory vesicles were observed in cytoplasm under the electron microscope. PM was noticed to be dissolved by chitinase. SDA-PAGE map showed that there were 7 clearer electrophoresis bands and their molecular weight was mainly distributed in more than 40 ku. Results from LC-ESI-MS/MS analysis showed that the PM proteins included sodium-potassium ATPase, ATP synthetase, actin and tubulin, thioredoxin, oxygenase, etc. These results indicated that the PM of E. sinensis is secreted by epithelial cells, improves absorption by forming compartment, and may participate in physiological processes of immune, osmotic regulation and nutrients transportation.