Water-soluble polysaccharides were isolated from a freshwater plant, Myriophyllum spicatum L., at elevated temperature and purified using DEAE Sepharose FF column to elucidate their structural properties and immunopotentiating effects. Polysaccharides were composed of neutral sugars (73.9-78.2%), sulfate esters (12.8-16.8%) and uronic acids (3.1-4.9%). Weight average molecular weight (M) of polysaccharides varied between 217.4 and 529.0 × 10 g/mol. Polysaccharide structures were predominantly consisted of glucose (31.9-59.1%) and galactose (16.6-34.5%) with lower amounts of mannose, xylose, arabinose and rhamnose. As the most active fraction, MSF1 induced RAW264.7 murine macrophage cells to release nitric oxide and initiate the expression of TNF-α, IL-1β, IL-6 and IL-12 cytokines. Besides, MSF1 activated NK-92 cells to produce TNF-α and INF-γ cytokines and initiate cell-mediated cytotoxicity via perforin/granzyme B, NKG2D receptor and FasL pathways. Stimulation of RAW264.7 and NK-92 cells with MSF1 resulted in high expressions of phosphorylated NF-κB, ERK, JNK and p38 proteins revealing the involvement of NF-κB and MAPKs signaling pathways. MSF1 possessed a highly-branched structure consisting a combination of (1 → 4)-Glcp, (1 → 6)-Glcp, (1 → 4,6)-Glcp, (1 → 3,4)-Galp and (1 → 2,3)-Galp residues. Altogether, the present findings suggested the potential application of sulfated-polysaccharides from M. spicatum L. for immunostimulation and thus the necessity for further biomedical research.