Cordyceps cicadae is a medicinal fungus popularly used in traditional Chinese medicine for treating cancer, asthma, and kidney diseases. In this study, crude polysaccharides (CP) and water-soluble nondigestible polysaccharides (NDPs) were prepared from the fruiting bodies of cultivated C. cicadae, and their physicochemical properties and anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages were examined. The results showed yields of CP and NDP of 3.42% and 1.17%, respectively. CP and NDP showed a similar monosaccharide composition, of which the predominant monosaccharide was mannose, followed by galactose and glucose. Differences in molecular weight distribution between CP and NDP were apparent; CP possessed two major (3.1 kDa and 21.5 kDa) and one minor (678.2 kDa) macromolecular populations, whereas NDP contained only one macromolecular population (24.4 kDa). Furthermore, CP but not NDP had a triple helix conformation. The bioassay results showed that, although both CP and NDP possess anti-inflammatory activity, NDP had stronger inhibitory effects on nitric oxide, IL-1β, and TNF-α production in LPS-stimulated RAW264.7 macrophages. From this study, we conclude that the differences in conformation and molecular weight distribution between CP and NDP may contribute to their differences in anti-inflammatory activity.