Intestinal microbes are believed to be involved in the pathogenesis of inflammatory bowel disease. Microbes and their products are generally well tolerated by intestinal epithelial cells in the intestinal tract of healthy individuals. It is of significance to understand what breaks down the established tolerance leading to intestinal barrier dysfunction and intestinal inflammation. T84 monolayer transported peptidoglycan (PGN) was determined by enzyme-linked immune assay. Mast cell line HMC-1 cell activation in response to PGN stimulation was observed with electron microscopy and measurement of histamine release. T84 monolayer barrier function was determined by recording the transepithelial electric resistance (TER) and measuring the permeability in response to PGN-induced HMC-1 cell activation. Expression of Toll-like receptor (TLR) 2 and nucleotide-binding oligomerization domain (NOD) 2 were determined by immunocytochemistry, real-time reverse transcription (RT)-PCR and Western blot. Exposure to PGN alone did not alter TER and permeability of T84 monolayers. T84 monolayers transported PGN from the apical chamber to the basal chamber of transwell system. TLR2 expressed on the surface of HMC-1 cells. HMC-1 cells absorbed PGN. HMC-1 cells released histamine in response to the PGN stimulation, which was blocked by pretreatment with antibodies or small interfering RNA against TLR2 or NOD2. In a co-culture system, T84 monolayer transported PGN activated HMC-1 cells and increased the horseradish peroxidase flux. TLR2 mediated the PGN-absorption in HMC-1 cells. Blockade of TLR2 or NOD2 abolished PGN-induced HMC-1 cell activation and T84 monolayer barrier dysfunction. T84 monolayer transported PGN activates HMC-1 cells to release chemical mediators to induce T84 monolayer dysfunction that are mediated by TLR2 and NOD2.