Stachyose is a prebiotic that traditionally extracted from plants, such as vegetable. It has been demonstrated to alleviate intestinal inflammation by regulating gut microbiota, but the mechanism has been unclear. This research aims to detect the potential mechanism of stachyose in alleviating the severity of ulcerative colitis (UC). The results indicated that the administration of stachyose could recover the body weight, protect against the colonic tissue damage, reduce the pro-inflammatory cytokines levels, and reverse the histological abnormalities in UC mice. Oral stachyose could restore dextran sulfate sodium (DSS)-induced disturbance in intestinal bacteria. Besides, the metabolome result of serum samples showed that stachyose treatment significantly altered serum metabolites against inflammatory responses in colitis mice. Also, a significant correlation can be found between 23 metabolite biomarkers and 18 differential genera. Our results provided a strong foundation for the future study of the protective role of stachyose in maintaining intestinal homeostasis.