Abstract: Objective: To investigate the protective effects of different doses of Fomes officinalis Ames polysaccharides (FOP) on intestinal flora and immune function in exercise-induced immunosuppression (EIS) rats. Methods: Fifty SPF male SD rats aged 7 to 8 weeks were randomly divided into control group (NC group), exercise training group (Ex group), exercise training+low-dose FOP group (LFOP group, 40 mg/(kg·d)), exercise training+medium-dose FOP group (MFOP group, 60 mg/(kg·d)) and exercise training+high-dose FOP group (HFOP group, 80 mg/(kg·d)). Ex group and FOP group were trained on the treadmill for 6 weeks to construct EIS model. LFOP, MFOP and HFOP groups were given FOP after each training. After the intervention, serum IgG, IgM, IL-6, IL-10, INF-γ, TNF-α, LPS levels and intestinal short-chain fatty acid content were detected by enzyme-linked immunosorbent assay. 16S rDNA was used to detect the structural changes of intestinal flora. The number of serum CD⁴⁺ and CD⁸⁺ was detected by cell analyzer. Western blot was used to detect the protein expression of ZO-1, Occludin, Claudin4 in the colon tissue. Results: Compared with Ex group, serum IgG, IgM, INF-γ, TNF-α, CD⁴⁺ and CD⁸⁺ of rats in LFOP, MFOP and HFOP groups were significantly increased (P<0.05), while serum levels of IL-6, IL-10 and LPS were significantly decreased (P<0.05). Intestinal short-chain fatty acid content and the abundance of Firmicutes, Bacteroidetes, verrucobacteria and Lactobacillus, Bacteroidetes, Spirillum (not classified) and Ekmanella were significantly increased (P<0.05); The expression of ZO-1, Occludin, Claudin4 proteins in colon tissue was significantly up-regulated (P<0.05). In addition, spleen index, thymus index, intestinal flora alpha diversity in MFOP and HFOP groups were significantly increased compared with Ex group (P<0.05). Conclusion: FOP supplementation may alter immune function, intestinal flora structure and intestinal barrier function in EIS rats.