The gut microbiota plays a central role in maintaining human health and has been linked to many gastrointestinal diseases such as ulcerative colitis (UC). Agaricus bisporus is a famous edible mushroom, and Agaricus bisporus polysaccharides (ABPs) and the two purified fractions (ABP-1 and ABP-2) were demonstrated to exhibit immunomodulatory activity in our previous study. Herein, we further found that ABPs, ABP-1, and ABP-2 possessed therapeutic effects against dextran sodium sulfate (DSS)-induced colitis in mice. ABPs, ABP-1, and ABP-2 could relieve body weight loss, colon atrophy, and histological injury, increase tight junction proteins, restore gut-barrier function, and inhibit inflammation. ABP-2 with a lower molecular weight (1.76 × 104 Da) showed a superior therapeutic effect than ABP-1 with a higher molecular weight (8.86 × 106 Da). Furthermore, the effects of ABP-1 and ABP-2 were microbiota-dependent, which worked by inducing Norank_f__Muribaculaceae and Akkermansia and inhibiting Escherichia-Shigella and Proteus. In addition, untargeted fecal metabolomic analysis revealed distinct modulation patterns of ABP-1 and ABP-2. ABP-1 mainly enriched steroid hormone biosynthesis, while ABP-2 significantly enriched bile secretion and tryptophan metabolism. In summary, ABPs, especially low-molecular-weight fraction, represent novel prebiotics for treatment of inflammatory gastrointestinal diseases.