The objective of this study was to evaluate the changes in some bacterial species of colonic microbiota, the clinical signs and the intestinal changes in mice with 2,4,6trinitrobenzenesulfonic acid (TNBS)-induced colitis. CD-1 male mice were randomly divided into three groups and inoculated intrarectally with saline, ethanol or TNBS solutions. Ethanol and TNBS treatments induced weight loss accompanied by mild and severe inflammation of the colon mucosa, respectively. However, TNBS-treated mice displayed significant differences compared to the saline group in terms of disease activity index and histological scoring. Both ethanol and TNBS groups showed an increased prevalence of Escherichia coli and Clostridium spp., a decrease in Lactobacilli and Bifidobacteria counts, as well as changes in the relative proportions of bacteria in the colon. The results confirm the validity of TNBS treatment to study the mechanisms involved in the pathogenesis and progression of inflammatory bowel diseases (IBD) in CD-1 mice. Gut microbiota may become a diagnostic biomarker with therapeutic potential for IBD in the future.