In this study, we aimed to investigate the immunomodulatory effects of polysaccharides from Cyclocarya paliurus leaves after acetylation modification (Ac-CPP0.1) on dendritic cells (DCs) and immunosuppressed mice. In vitro, Ac-CPP0.1 promoted phenotypic and functional maturation of DCs. Specifically, it increased the expression of costimulatory molecules (CD80, CD86, and MHC II) and the secretion of cytokines (TNF-α, IL-6, IL-1β, IL-10, IL-12p70) of DCs. In vivo, Ac-CPP0.1 significantly improved immunosuppression of mice, which was manifested by increased body weight and immune organ index, up-regulated cytokines (IL-4, IL-17, TGF-β3, and TNF-α), and restored short-chain fatty acid (SCFAs) levels of intestinal. The immunoactivation of Ac-CPP0.1 in DCs and in mice is linked to the activation of the TLR4/NF-κB signaling pathway. Furthermore, Ac-CPP0.1 reversed intestinal flora imbalance caused by cyclophosphamide. At the species level, Ac-CPP0.1 increased the abundance of unclassified_Muribaculaceae, unclassified_Desulfovibrio, Bacteroides_acidifaciens and Faecalibaculum_rodentium, decreased the level of Lactobacillus_johnsonii, unclassified_g_Staphylococcus and Staphylococcus_nepalensis. In summary, Ac-CPP0.1 has considerable immunomodulatory potential, which is beneficial to the future utilization and development of Cyclocarya paliurus.