Background and Aims: Intestinal microbiota is required to maintain immune homeostasis and intestinal barrier function. At the same time, intraluminal bacteria are considered to be involved in inflammatory bowel disease and are required for colitis induction in animal models, with the possible exception of dextran sulphate sodium DSS colitis. This study was carried out to ascertain the mechanism underlying the induction of colitis by DSS in the absence of bacteria. Methods: Conventional and germ-free GF Naval Medical Research Institute NMRI mice were used, plus conventional mice treated with an antibiotic cocktail to deplete the intestinal microbiota ‘pseudo-GF’ or PGF mice. The differential response to DSS was assessed. Results: Conventional mice developed DSS-induced colitis normally, whereas GF mice showed only minimal inflammation no colonic thickening, lower myeloperoxidase activity, IL-6, IL-17, TNF-α, and IFN-γ secretion by splenocytes and mesenteric cell cultures, etc.. However, these mice suffered enhanced haemorrhage, epithelial injury and mortality as a consequence of a weakened intestinal barrier, as shown by lower occludin, claudin 4, TFF3, MUC3, and IL-22. In contrast, PGF mice had a relatively normal, albeit attenuated, inflammatory response, but were less prone to haemorrhage and epithelial injury than GF mice. This was correlated with an increased expression of IL-10 and Foxp3 and preservation barrier-related markers. Conclusions: We conclude that enteric bacteria are essential for the development of normal DSS-induced colitis. The absence of microbiota reduces DSS colonic inflammation dramatically but it also impairs barrier function, whereas subtotal microbiota depletion has intermediate effects at both levels.