A new polysaccharide (cDFP-W1) with high immunostimulatory activities was isolated from the stems of Dendrobium fimbriatum Hook. The analysis of the physicochemical properties showed that cDFP-W1 consisted of mannose and glucose in a molar ratio of 1 : 3.84, and its relative molecular weight was 4.0 × 104 Da. Structural analysis implied that the linear backbone of cDFP-W1 was composed of α-1,4-d-Glcp, β-1,4-d-Manp, 3-O-acetyl-α-1,4-d-Glcp and α-1,4,6-d-Glcp, and its branches were the terminal β-d-Manp that was attached to the C-6 position of α-1,4,6-d-Glcp. An in vivo immunostimulatory assay exhibited that cDFP-W1 at 200 mg kg-1 could significantly increase the proportions of CD4+ T-cell subpopulations, B cells, natural killer cells and dendritic cells, decrease the proportion of CD8+ T-cell subpopulations, and upregulate the percentage of activated macrophages (p < 0.01) in the spleen of mice. An in vitro immunostimulatory assay revealed that cDFP-W1 could effectively promote the proliferation of spleen lymphocytes, enhance the proliferation and phagocytosis of macrophage RAW264.7 cells, and stimulate the mRNA expression and extracellular release of NO, TNF-α and IL-1β of RAW264.7 cells. The western blot experiment suggested that the immunostimulatory activities of cDFP-W1 were closely related to the activation of MAPKs, NF-κB and PI3K/Akt signaling pathways.