Ulcerative colitis (UC) is a chronic gastrointestinal disease whose incidence is increasing rapidly worldwide. Anti-inflammatory medications, including 5-aminosalicylic acid (5-ASA), corticosteroids, and immunosuppressants, are used for its treatment; however, new alternatives would be required due to the serious side effects of some of these medications. N-Acetylglucosamine (NAG) is an amino sugar composed of mucin that is secreted by intestinal epithelial cells. It is also used to promote the growth of intestinal bacteria. The current study aimed to determine the efficacy of NAG against dextran sulfate sodium (DSS)-induced chronic colitis and elucidate its mechanism of action. Mice were randomly divided into control, DSS, 0.1% sulfasalazine, 0.1% NAG, 0.3% NAG, and 0.3% NAG-dimer (NAG-D) groups, and results showed that colitis-induced body weight loss, disease activity, colonic tissue damage, colon length shortening, and the loss of mucin-secreting area were significantly improved in the NAG-D group. The intestinal permeability indicator, serum CD 14 level, and expression of the tight junction protein, occludin, were both improved in the 0.3% NAG group. Inflammatory biomarkers, including GATA3, IFN-γ, p-IκBα, COX2, TGF-β1, and Smad7, were significantly lower in the 0.3% NAG and NAG-D groups than in the DSS group. The intestinal microbial composition was most significantly altered in the 0.3% NAG group, showing decreased ratios of pathogenic bacteria, such as Betaproteobacteria, especially Burkholderiales. The results overall suggested that NAG or NAG-D supplementation can alleviate inflammation by strengthening the intestinal barrier function and maintaining gut microbiota homeostasis in a DSS-induced colitis mouse model.