This paper intends to find out the molecule marker of the progress of proteomics in colonic mucosa from sub-healthy people with constipation, so as to provide theoretical basis for the pathological changes of colonic mucosa in sub-healthy people with constipation. Two-dimensional electrophoresis (2-DE) was used to separate the total proteins of sub-healthy people with constipation and those of the healthy volunteers. ImageMaster 2D Elite soft was applied to analyze 2-DE images. Finally, the differential protein spots between the two groups were identified by peptide mass fingerprint (PMF), based on matrix-assisted laser desorption /ionization time of flight mass spectrometry (MALDI -TOF-MS) and searching in database. A reproducible 2-DE pattern in colonic mucosa of sub-healthy people with constipation and the healthy volunteers was established. There were 501.00±37.16 in the average gel of people with constipation, and 536.00±41.63 in the healthy volunteers. There were 46.00±7.82 differentially expressed protein spots in average between the two groups. Then 20 spots, that were significantly differentially expressed, were picked for identification, and 17 proteins were identified by mass spectrometry, in which seven decreased significantly while ten increased significantly. Some of those proteins participated in protein synthesis and degradation, or chaperone, or adjust oxidoreduction, while some were involved in signal transduction. Some differentially expressed proteins: β-actin, YWHAZ and PBP-Ⅰ (phosphatidylethanolamine- binding protein Ⅰ) were further confirmed by Western blot analysis and researched for clinical application. The proteomic expression in colonic mucosa of people with constipation is significantly different from that of the healthy volunteer controls. β-Actin and YWHAZ protein are down-regulated, and PBP-Ⅰ protein is up-regulated, which may be associated with the pathogenesis of constipation. Proper intervention on this stage can promote the transition from sub-healthy status to healthy status.