The biosynthesis and modification of mucopolysaccharides and glycosaminoglycans (GAGs), secreted from gastrointestinal mucosal cells, are increased in colitis and influence the viability of the defense barrier. Therefore, to evaluate the role of GAG-degrading intestinal microflora during the progression of colitis, we investigated the degradation activity of intestinal bacterial GAG, cytotoxicity of GAGs and their metabolites, such as iduronic acid, D: -uronic acid or D: -glucuronic acid and D: -galactosamine or D: -glucosamine, against intestinal cells. We also tested their deteriorative effects against colitis. Colitis was induced using 2,4,6-trinitrobenzene sulfonic acid (TNBS) with and without antibiotics in mice. The TNBS treatment caused colon shortening, increased myeloperoxidase activity, induced IL-1beta, TNF-alpha, and IL-6 expression in the colon, activated NF-kappaB, and potentiated the GAG-degrading activities of intestinal microflora. The antibiotic treatment inhibited colon shortening, decreased myeloperoxidase activity, and reduced proinflammatory cytokine expression, NF-kappaB activation, and GAG degradation, induced by TNBS. Among the GAG metabolites, d-glucosamine and d-galactosamine showed cytotoxicity against intestinal cells, Caco-2 and IEC-18 cells, synergistically deteriorated the cytotoxicity of TNBS as well as the TNBS-induced colitis in mice. Based on these findings, intestinal microflora may degrade GAGs in colitis, their metabolites deteriorate the progress of colitis and antibiotics ameliorate the colitis by the inhibition of GAG-degrading bacterial growth.