Background: Lespedeza cuneata (Dum. Cours.) G. Don, a perennial legume native to Eastern Asia, has been used therapeutically in traditional Asian medicine to protect the function of liver, kidneys and lungs. However, its effect on inflammatory nitric oxide (NO) production and the active constituents have not yet been explored. Objective: In this study, we investigated the phytochemical constituents of L. cuneata and evaluated their effect on NO production using lipopolysaccharide (LPS) stimulated BV2 cells. Materials and Methods: The 80% methanol extract of the aerial part of L. cuneata were used for the isolation of flavonoids. The isolated compounds were elucidated by various spectroscopic methods including nuclear magnetic resonance and mass spectrometry spectrometry. To evaluate the effect on inflammatory NO production, LPS stimulated murine microglia BV 2 cells were used as a screening system. Results: Nine flavonoids were isolated from the aerial parts of L. cuneata. Among the isolated flavonoids, compounds 4, 5, 7 and 9 are reported from the genus Lespedeza for the first time. Moreover, compounds 1 and 6 showed significant inhibitory effects on NO production in LPS stimulated BV2 cells without cell toxicity. Conclusion: In this study, nine flavonoids were isolated from L. cuneata. Among the compounds, only 1 and 6, which have free hydroxyl groups at both C3 and C7 showed significant inhibitory activity on NO production in LPS stimulated BV2 cells. These results suggested L. cuneata and its flavonoid constituents as possible candidate for the treatment of various inflammatory diseases.