Ulcerative colitis (UC) is a public health concern. The quest for drugs characterized by minimal side effects and optimal safety profiles for managing UC has emerged as a primary research topic. Recent evidence suggests the efficacy of Lactobacillus-derived exopolysaccharides containing mannose (MnEPSs) in alleviating UC. Lactobacillus could not synthesize MnEPSs in the glucose-only MRS medium, but with mannose added to the carbon source, MnEPSs were produced directionally, of which mannose accounts for 5.9–7.8% of the total sugar content. Subsequently, MnEPS-6M3, MnEPS-Q5M22, MnEPS-D4L1, and MnEPS-CCFM 1393 were used to investigate the mechanism of UC relief. The results showed that compared with non-MnEPSs, MnEPSs had the better effect on reliving UC. Phenotypically, four MnEPSs alleviated weight loss, DAI score, crypt disappearance, inflammatory cell infiltration, and mucosal edema of UC in mice. Mechanistically, MnEPSs increased the expression of ZO-1 by 52.38–115.87% to repair intestinal barrier, inhibited inflammatory factors (TNF-α, IL-1β, IL-6, IFN-γ and IL-17) and increasd anti-inflammatory cytokines (IL-10) by reducing colonic F4/80+ macrophages (decreased 4.34–7.01%) and colonic iNOS+ M1 macrophages (decreased 7.3–10.52%), and promoted the production of acetic acid (increased 153.21–226.93%), propionic acid (increased 360.71–528.57%), and butyric acid (increased 127.45–156.86%) in cecal contents. Interestingly, MnEPS-Q5M22 and MnEPS-CCFM 1393 increased the abundance of butyric acid-producing Dunaliella. In summary, this study provides a theoretical basis for the directional synthesis of functional MnEPSs.