Water soluble polysaccharides from seven batches of Gymnadenia conopsea were firstly investigated and compared using high performance size exclusion chromatography coupled with multi-angle laser light scattering/refractive index detector (HPSEC-MALLS/RID) and saccharide mapping based on polysaccharide analysis by carbohydrate gel electrophoresis (PACE), respectively. The results showed that the weight-average molecular weight (M-w) and the radius of gyration (< S-2 >(1/2)(z)) of polysaccharides were ranging from 4.46 x 10(5) to 7.41 x 10(5) Da and 73.3-94.2 nm, respectively. By applying the polymer solution theory, the exponent (v) values of < S2 >(1/2)(z) = kM(w)(v) were ranging from 0.36 to 0.42, which indicated that polysaccharides from G. conopsea existed as globular in the aqueous solution. Furthermore, the results showed that alpha-1,4- and beta-1,3(4)-glucosidic, alpha-1,5-arabinosidic, beta-1,4-mannosidic and alpha-1,4-D-galactosiduronic linkages existed in polysaccharides from G. conopsea. The similarity of the hydrolysates of polysaccharides in G. conopsea collected from different regions was high. Moreover, the nitric oxide released from RAW 264.7 cells induced by polysaccharides were significantly affected by their alpha-1,5-arabinosidic and beta-1,3(4)-glucosidic, especially alpha-1,4-D-galactosiduronic and beta-1,4-mannosidic linkages. These results are beneficial for better understanding of the structuresebioactivity relationship of polysaccharides from G. conopsea, and helpful to improve their pharmacological activity-based quality control.