Bioassay-guided investigation was performed to identify the active constituents from a methanol extract of Polygala japonica, a folk medicinal plant widely used in China to treat inflammatory diseases. The n-BuOH and EtOAc fractions of the P. japonica methanol extract, which show significant anti-inflammatory activity in in vivo test, were further subjected to column chromatography to afford six triterpene glycosides, marked here as saponins 1-6. All compounds were evaluated for their anti-inflammatory activity in the carageenan-induced mouse paw edema test, and saponins 1, 4 and 5 showed significantly anti-inflammatory effects on both phases of carageenan-induced acute paw edema in mice. Saponin 5 was also found to significantly inhibit the production of inflammatory mediators - nitric oxide (NO) in LPS-stimulated RAW264.7 macrophages, with no obvious effects on macrophage viability.