This study examines the ameliorative effects of lemon (Citrus limon) peel (LP) powder on intestinal inflammation and barrier defects in dextran sulfate sodium (DSS)-induced colitic mice. The whole LP powder was fractionated into methanol (MetOH) extract and its extraction residue (MetOH residue), which were rich in polyphenolic compounds and dietary fibers, respectively. Mice were fed diets containing whole LP powder, MetOH extract, and MetOH residue for 16 d. DSS administration for 9 d induced bodyweight loss, reduced colon length, reduced the colonic expression of tight junction proteins including zonula occludens-1 and -2, and claudin-3 and -7, and upregulated colonic mRNA expression of interleukin 6, chemokine (C-X-C motif) ligand 2, and C-C motif chemokine ligand 2. Feeding LP powder restored these abnormalities, and the MetOH residue, but not MetOH extract, also showed similar restorations. Feeding LP powder and MetOH residue increased fecal concentrations of acetate and n-butyrate. Taken together, LP powder reduced intestinal damage through the protection of tight junction barriers and suppressed an inflammatory reaction in colitic mice. These results suggest that acetate and n-butyrate produced from the microbial metabolism of dietary fibers in LP powder contributed to reducing colitis.