Three water-soluble polysaccharides (AMAP-1, AMAP-2 and AMAP-3) were isolated and purified from Atractylodis Macrocephalae Rhizoma by using the combination of ion-exchange chromatography and gel permeation chromatography. The structures of the polysaccharides were characterized by chemical derivatization, HPGC, GC–MS, FT–IR, and NMR techniques. Structural analyses show that the three polysaccharides are pectin-type macromolecules consisting of homogalacturonan (HG) and rhamnogalacturonan type I (RG-I) regions in different ratios. Immunostimulatory assay highlighted that the RG-I-rich AMAP-1 and AMAP-2 with high molecular weights can stimulate RAW264.7 macrophages to release nitric oxide, but HG-rich AMAP-3 with a low molecular weight cannot. This finding suggests that the immune activity may be related to the side chains of the RG-I region, which provides a certain theoretical guidance for further exploring the structure–activity relationship. Meanwhile, AMAP-1 and AMAP-2, especially AMAP-2, from Atractylodis Macrocephalae Rhizoma show potential as immune adjuvants. • Three purified native polysaccharides, namely, AMAP-1, AMAP-2 and AMAP-3 were isolated from AMR. • Structure of three polysaccharides was characterized. • The RG-I-rich AMAP-1 and AMAP-2 with higher molecular weights can stimulate RAW264.7 macrophages to release NO. [ABSTRACT FROM AUTHOR]