Ulcerative colitis (UC) is a chronic inflammatory bowel disease with inadequate therapeutic options and has a significant impact on patient quality of life. We optimized the preparation of agarotriose (A3), a functional oligosaccharide, and assessed its health-promoting potential in a dextran sulfate sodium-induced UC mouse model. The beneficial effects of A3 were evaluated through histopathological, biochemical, immunological, and transcriptomic analyses. The medium dose (200 mg/kg) exhibited the greatest protective effect. A3 effectively restored substantial gut barrier integrity by dose-dependently increasing goblet cell numbers and enhancing the mucus layer thickness through MUC2 upregulation. It also re-established the continuous distribution of tight junction proteins (claudin-1, occludin, and ZO-1), thereby reinforcing gut barrier function. Biochemically, A3 mitigated oxidative stress by reducing malondialdehyde and myeloperoxidase levels, while enhancing antioxidant defenses by boosting total antioxidant capacity and total superoxide dismutase activity. Immunologically, A3 notably suppressed pro-inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, and IL-6) and increased anti-inflammatory IL-10 levels, promoting a protective immune response. Transcriptomic profiling identified 3871 differentially expressed genes, with A3 downregulating the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway horizontal line a key regulator of inflammation and cell survival. This was further confirmed by reduced PI3K, Akt, and mTOR phosphorylation, verifying pathway suppression.