Dictyosphaerium chlorelloides is a green microalga from the Chlorella clade that produces highly viscous exocellular polysaccharides. The cell wall polysaccharides of this alga have not been studied in detail. In this article, water-soluble polysaccharides from D. chlorelloides biomass were extracted with hot water and purified by preparative chromatography. The composition, structural features and molecular masses of subsequently eluted fractions F1, F2, F3, F4 and F5 (minor) were determined. Three high-yield products F1, F3 and F4 consisted mainly of galactopyranosyl, 2-O-methyl-galactopyranosyl, rhamnopyranosyl and mannopyranosyl units at different proportions, while F2 was rich in glucose. Immunoactivity of these fractions was evidenced in a mixed population of immune cells derived from mice spleens after incubation with polysaccharides by flow cytometry, MTT and Immunospot assays. These fractions, except F2, demonstrated selective immunostimulant activity, and the F1 fraction induced the most potent effect, closely followed by the F3 and F4 fractions. The in vivo mechanism of their action is associated with the activation of innate immunity and shapes the immune response to the Th1 type.