Objective: To investigate the altations of proteins in the colonic mucosa of chronic slow transit constipation (STC) rats with a 2-DE-based proteomic method and analyze the function of these down-regulated proteins so as to provide theoretical basis for the pathogenesis of intestinal mucosa of chronic STC rats. Methods: STC model was established by feeding rats with 8 mg/(kg·d) diphenoxylate for 120 d. An experimental model of chronic STC rat was used for separation of proteomics from colonic mucosa using two-dimensional electrophoresis (2-DE). Proteins altered in expressional level were identified by Image Master 2DElite, mass spectrometry, and bibliometrics were applied to identify the differential protein expression and their clinical significance and function were analyzed. Results: Obvious differential protein expression was observed in the pathogenesis of STC, including mast cell protease (Al), non-specific dipeptidase (A2) and chondrosome succinate dehydrogenase precursor (A3). The expressions of Al, A2 and A3 were down-regulated in the gel graph of STC rats. Conclusion: The down-regulation of chondrosome succinate dehydrogenase, mast cell protease as well as non-specific dipeptidase in rat colon suggests the functional impairment of the oxidoreduction of mitochondrion is very important in the genesis and development of STC. The immunological reaction of STC rats is weakened, and the function of digesting and absorbing protein may be damaged to some extent.