The chemical constituents were separated from Hedyotis diffusa Willd by using silica gel,Sephadex LH-20,preparative liquid chromatography and other chromatographic separation methods.The obtained compounds were identified by NMR and MS.Thirteen compounds isolated from H. diffusa include pentadecanoic acid heptadecyl ester (1),stigmasterol (2),2-hydroxy-1-methoxy-3-methylanthraquinone (3),7-hydroxytectoquinone (4),ursolic acid (5),oleanolic acid (6),2-butyl-1-hydroxyanthra-9,10-quinone (7),betulinic acid (8),rutin (9),quercetin (10), trans-p-hydroxycinnamic acid (11), trans-p-methoxycinnamic acid (12) and kaempferol (13).Compounds 1,4 and 7 were isolated from H. diffusa for the first time.CCK-8 was used to detect the inhibitory effects of different polar sites and compounds on the proliferation  of A549 cells in vitro.The chloroform extract and ethyl acetate extract from H. diffusa both inhibited the proliferation of A549 cells,also showed significant dose-effect relationship.When the concentrations of extract was from 25 μmol/L to 400 μmol/L,the inhibition rate of cell proliferation for the chloroform and ethyl acetate sites increased from 12.9%,43.32% to 80.49%,63.74%,respectively.Ursolic acid,oleanolic acid and betulinic acid can significantly inhibit the proliferation of A549 cells with IC₅₀ values of 12.80,2.81 and 8.38 μmol/L,respectively.