Arctiin (I), arctigenin (II), matairesinol (III), and lappaol F (IV) were isolated and purified from the traditional Chinese medicine Fructus Arctii by high-speed counter-current chromatography (HSCCC). The crude extracts from Fructus Arctii were treated with D101 macroporous resin first and divided into two parts: fraction 1 and fraction 2. Fraction 1 was separated by ethyl acetate-n-butanol-water (4:0.5:5, v/v/v) and yielded 164mg of I from 250mg of fraction 1. Fraction 2 was separated by n-hexane-ethyl acetate-methanol-water (2:3:2:3, v/v/v/v) and yielded 27mg of II, 5mg of III, and 3mg of IV from 150mg of fraction 2. The purities of the four compounds were 99.64%, 98.48%, 96.16%, and 91.41%, respectively, as determined by HPLC-DAD. The chemical structures of the isolated compounds were identified by MS, UV, H-1 NMR, and C-13 NMR analysis.