Background. Inflammatory bowel disease (IBD) is a chronic relapsing disorder of the gastrointestinal tract. The nutrition care gut relief formula (GR), a combination of natural products and nutrients, has been shown to benefit gastrointestinal health. However, the underlying mechanism responsible for this effect is incompletely defined. Objective. This study was conducted to evaluate the hypothesis that GR could attenuate dextran sulfate sodium (DSS)-induced colitis by enhancing intestinal mucosal immunity and regulating intestinal microflora in mice. Methods. Six-week-old C57BL/6J mice orally administered with GR (7.5 mg per mouse per day) or an equal volume of vehicle were treated with sterile water or 2.5% DSS for 6 days to induce colitis. Histological damage, inflammatory cell infiltration, and colonic microbiome community were analyzed to evaluate the beneficial effect of GR. Results. GR administration ameliorated the severity of colitis as evidenced by reduced body weight loss, decreased colon shortening, reduced myeloperoxidase (MPO) activity, inhibited proinflammatory cytokine secretion, and decreased histological damage in DSS-challenged mice. Additionally, enhancement of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) in response to DSS was attenuated by GR administration. Meanwhile, DSS treatment resulted in reduction of the glutathione (GSH) level and tight junction protein abundance, as compared with the controls. Of note, these adverse effects were remarkably eliminated by GR administration. Further study showed that the protective effect of GR was associated with the inhibited activation of STAT3 and NF-κB signaling pathways, as well as upregulated abundances of Lactobacillus in the colon tissues of mice. Conclusion. Collectively, the data provided herein demonstrated that GR administration alleviated intestinal mucosal inflammation and mucosal barrier dysfunction. These beneficial effects were associated with inhibited activation of STAT3 and NF-κB signaling pathways, as well as upregulated abundances of Lactobacillus in the colon tissues of mice.