This study aims to investigate the chemical constituents and inhibitory effect on neuroinflammation of Eclipta prostrata. D101 macroporous resin,silica gel,Sephadex LH-20,and semi-prepared high-performance liquid chromatography were used to isolated and purified the chemical constituents of 95% ethanol extract Eclipta prostrata. Twenty monomer compounds were identified as 3,4-seco-olean-12-en-4-ol-3,28-dioic acid (1),echinocystic acid (2),eclalbasaponin Ⅱ(3),eclalbasaponin I (4),ecliptasaponin B (5),eclalbasaponin Ⅳ (6),eclalbasaponin Ⅵ (7),eclalbasaponin Ⅶ(8),orobol (9),apigenin (10),cirsimaritin (11),nobiletin (12),luteolin-7-O-β-D-glucoside (13),aurantiamide acetate (14),plantagoguanidinic acid (15),plumbagine B (16),wedelolactone (17),pinoresinol (18),α-formylterthienyl (19),and α-terthienylmethanol (20) based on the nuclear magnetic nuclear resonance (NMR) data and comparison with corresponding data of the reported literatures.Compounds 1,14,and 15 were isolated from E. prostrata for the first time.Some of the isolated compounds were assessed for their inhibitory effects on neuroinflammation by detecting NO level in BV2 cells induced by lipopolysaccharide (LPS),compound 15 significantly reduced NO release in LPS-induced BV2 cells with an IC₅₀ of 37.3 μmol/L and showed no cytotoxicity.Guanidine alkaloid 15 was first reported with anti-inflammatory activity in LPS-induced BV2 cells.