Abstract: Pueraria lobata (Willd.) Ohwi. was extracted for two times with 70% ethanol and the 70% ethanol-extracts was condensed. Various column chromatography with AB-8 macroreticular resin, Toyopearl HW-40, pharmadex LH-20, and silica gel were employed for the isolation and purification of the 70% ethanol-extracts from Pueraria lobata (Willd.) Ohwi. Five compounds were isolated and their structures were identified by physiochemical properties and spectral analysis (UV, IR, MS, ¹H NMR, ¹³C NMR, HMQC, HMBC, etc.): (4R)-3-［2-hydroxy-4-methoxyphenyl］-4-(4-β-D-glucopyranosyloxybenzyl) but-2-en-4-olide (1), 4′,8-dimethoxyl-7-O-β-D-glucopyranosyl isoflavone (2), eicosanoic acid (3), hexadecanoic acid (4), tetracosanoid acid-2,3-dihydroxypropyl ester (5). Compound 1 was a new compound, and compounds 2, 3, 4 were isolated from this plant for the first time.