Two neutral polysaccharides named POP-1 and PCP-1 were purified from Polygonatum odoratum and P. cyrtonema, respectively. Results showed that both of them were composed of fructose and glucose, and the average molecular weights of them were about 5 kDa. FT-IR, 1D- and 2D-NMR analyses revealed that both POP-1 and PCP-1 contained a (2→1)-linked β-d-fructofuranose (Fruf) backbone and (2→6)-linked β-d-Fruf side chains with an internal α-d-glucopyranose (Glcp) in neokestose form. The difference between these two purified polysaccharides were that PCP-1 possessed of an acetyl group attached at O-3 of α-d-Glcp residue. In addition, PCP-1 exhibited a little better immune stimulating activity than POP-1 on cell viability and IL-6 production of RAW 264.7 macrophages. These results indicated that the acetyl group might affect the immunoregulatory activity and PCP-1 have more potential to be explored as an immunomodulatory agent.