The purpose of this study was to investigated the potential role of gut microbiota in protecting the intestinal barrier and improving nutritional metabolism in 5-FU-treated S180 tumour-bearing mice after treatment with oyster polysaccharide (CHP). CHP, with an α-(1→4) d-linked glucose backbone and (→4,6)-α-d-Glc-(1→) branches every 4.7 residues on average, increased the villus height, crypt depth, mucosa thickness, villus surface area and V/C ratio; decreased the expression of IL-1β, IL-6, and TNF-α; and even restored the TP, ALB, PA, TRF, IgA, IgM and IgG levels to normal levels. All these factors are related to CHP increasing the propionic acid- and butyric acid-producing microorganisms and decreasing the production of Bacteroides, Prevotellaceae_UCG-001 and Rikenellaceae_RC9_gut_group, thus affecting the TLRs signalling pathway. In conclusion, CHP attenuates 5-FU-induced intestinal mucositis and malnutrition by regulating gut microbiota, and can improve the prognosis of patients receiving chemotherapy.