Inflammatory bowel disease (IBD), a recurrent gastrointestinal disease, is characterized by dysbiosis and inflammation. 2'-fucosyllactose (2'-FL) shows benefits in maintaining gut microenvironment homeostasis. This study aims to investigate the protective mechanism of 2'-FL against experimental IBD using integrated analysis of colonic proteomics and microbiota 16S rRNA sequencing technologies. A murine model of experimental colitis was induced by dextran sodium sulfate (DSS) for 7 days (day 0-day 7). 2'-FL (250 mg/kg/d) was orally administered for 7 days. On day 7, all mice were sacrificed and their colon tissues were collected for morphological examination and label-free quantitative proteomics analysis. The fecal samples were collected for microbiota 16S rRNA sequencing. 2'-FL significantly ameliorated DSS-induced pathological damage and restored the epithelial integrity of colon tissues in mice. Colonic proteomics and GO analysis showed 681 differentially expressed proteins (DEPs) in response to 2'-FL administration. These DEPs were mainly enriched to GO terms of response to bacterium and acute-phase response. In addition, 2'-FL reversed the compensatory increase of haptoglobin, serpina3n, Arg2 and Reg3. It also reduced the accumulation of pro-oxidant indicator Cyp2e1, in the colon of colitis mice. Moreover, 2'-FL ameliorated colonic dysbiosis by suppressing the overgrowth of pathogenic Proteobacteria and reversed the reduction in abundance of prebiotic bacterial genus including Lachnospiraceae_NK4A136_group. Notably, the expression of Arg2 and Cyp2e1 showed strong correlation; Reg3b and Reg3g were significantly associated with Lactobacillus. 2'-FL prevented DSS-induced intestinal inflammatory damage through exerting prebiotic capacity and regulating the intestinal immune response to gut microbiota.