Physalis alkekengi L. (PM) is a traditional Chinese medicine with valuable nutritional and medicinal potential. Polysaccharides were considered as the important active component of calyx of P. alkekengi (PMC). In this study, four polysaccharides (PMCPs) were successfully isolated and purified by extraction, deproteination, dialysis and column chromatography. The structure characterization showed that PMCPs were mainly composed of mannose, glucosamine, ribose, rhamnose, glucose, sorbose, glucuronic acid and galacturonic acid with the molecular weights of 16.27, 71.53, 77.17 and 299.45 kDa. The FT-IR and NMR analysis suggested the presence of α and β configurations of PMCPs. PMCPs have the triple helix conformation which was determined by Congo red analysis. Moreover, the potential in vitro anti-inflammatory activity of PMCPs was investigated in lipopolysaccharide-treated RAW264.7 cells. The results indicated that PMCPs could significantly inhibit NO production at the experimental concentrations. S-PMCP-3 was found to decrease the production of pro-inflammatory cytokines including PGE2, TNF-α, IL-1β and IL-6. The findings indicated that PMCPs may be a promising natural source for addressing inflammatory damage.