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Hemerocallis citrina Baroni (H. citrina) has been commonly utilized in diet and conventional medicine. In this study, 21 compounds were obtained from the flower buds of H. citrina. Compounds 1, 2 and 3 were three novel compounds isolated from Hemerocallis genus. Compounds 4, 6, 7, 9, 10, 13–19 were isolated from Hemerocallis genus for the first time. It indicated that compounds 1, 2, 3, 4, 5, 7 and 13 exhibited a significant NO inhibitory effect in the LPS induced BV2 cells. The analysis result of PPI and molecular docking showed that compound 1 may play an anti-neuroinflammatory role through MAPK signaling pathway. The result of pharmacological experiments proved that compound 1 effectively inhibited the generation of NO and inflammatory cytokines by inhibiting the activation of MAPK and NF-κB pathway. This research provided a basis for further study and utilization of H. citrina in anti-neuroinflammatory effect.