OBJECTIVES: Diarrhea-predominant irritable bowel syndrome (IBS-D) patients show altered epithelial permeability and mucosal micro-inflammation in both proximal and distal regions of the intestine. The objective of this study was to determine the molecular events and mechanisms and the clinical role of upper small intestinal alterations. METHODS: Clinical assessment and a jejunal biopsy was obtained in IBS-D patients and healthy subjects. Routine histology and immunohistochemistry was performed in all participants to assess the number of mast cells (MCs) and intraepithelial lymphocytes. RNA in tissue samples was isolated to identify genes showing consistent differential expression by microarray analysis followed by pathway and network analysis in order to identify the biological functions of the differentially expressed genes in IBS-D. Gene and protein expression of tight junction (TJ) components was also assessed by quantitative real-time polymerase chain reaction and confocal microscopy to evaluate the pathways identified by gene expression analysis. RESULTS: The analysis reveals a strong association between the transcript signature of the jejunal mucosa of IBS-D and intestinal permeability, MC biology, and TJ signaling. The expression of zonula occludens 1 (ZO-1) was reduced in IBS-D at both gene and protein level, with protein redistribution from the TJ to the cytoplasm. Remarkably, our analysis disclosed significant correlation between ZO proteins, MC activation, and clinical symptoms. CONCLUSIONS: IBS-D manifestations are linked to molecular alterations involving MC-related dysregulation of TJ functioning in the jejunal mucosa.