The structural characteristics and immunoregulatory activities of neutral heteropolysaccharide (AVRP-N) separated from the roots of Apocynum venetum L. were extensively investigated. The results showed that the weight average molecular mass (Mw) of AVRP-N was 6.430 x 103 Da. Moreover, the backbone is composed of natural acetylated (1 -> 4)-beta-D-Man and (1 -> 5)-alpha-L-Ara domains. The mannan is composed of -> 4)-beta-D-Manp- (1 ->, -> 4)-beta-D-Glcp-(1 ->, and the terminal group alpha-D-Galp-(1 -> attached to -> 4,6)-beta-D-Manp-(1 -> at O-6. Araban is composed of -> 5)-alpha-L-Araf-(1 ->; the terminal group alpha-L-Araf-(1 -> attached to -> 2,3,5)-alpha-L-Araf-(1 -> at O-2, O-3 and -> 3,5)-alpha-L-Araf-(1 -> at O-3. In addition, the senior structure shows that AVRP-N has a triple-helix confor-mation. Furthermore, AVRP-N exhibited immunomodulatory effects, which could significantly regulate the proliferation of mouse splenic lymphocytes by enhancing the secretion of the cytokines (IFN-gamma, IL-2, IL-4, and IL -10). Our results provide new structural and immunoregulatory information for natural polysaccharides derived from Apocynum venetum L.