To explore the pathogenesis of irritable bowel syndrome (IBS) and the regulation of the network relationship between differential proteins. Sixteen female SD rats of clean grade were randomly divided into IBS group (n = 8) and control group (n = 8). A rat model of IBS was established by a special odor of mothball as a conditional stimulation and colorectal distension plus the classic conditions of physical restraint as a non-conditional stimulation in turn. The total proteins were extracted from colon mucosa of two different rat groups. After preparation of total proteins, solubilized proteins were separated by two-dimensional differential gel electrophoresis. Those differential protein spots were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The identified proteins were classified through an online web gene ontology tool. Some of the valuable differently expressed proteins were chosen for further verification by Western blotting. Intensity changes of 19 spots were detected among 1396 protein spots. A total of 13 different proteins were identified by MALDI-TOF MS successfully; eight of these were up-regulated and five down-regulated in colon mucosa of IBS rat. The eight over-expressed proteins were cytokeratin 8, protein disulfide isomerase A3 (PDIA3), peroxiredoxin-6, cathepsin S, heterogeneous nuclear ribonucleoprotein F, eukaryotic translation initiation factor, carbonyl reductase 1, glyoxalase I; five under-expressed proteins were alpha-enolase, transgelin, serpinB5, cardiac alpha-actin 1 and 40S ribosomal protein SA. The results of Western blotting confirmed that PDIA3 was indeed over-expressed in colonic mucosa of IBS rat. Some proteins related to immunity of intestinal tract, inflammation and nerve are differently expressed in colonic mucosa of IBS rat. It is suggested that immunity, inflammation and neuro-endocrine network in gut are associated with the pathogenesis of IBS.