A preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of ethyl acetate-n-butanol-water (2:7:9, v/v/v) was successfully performed to isolate and separate clemastanin B and indigoticoside A from the plant of Radix Isatidis, a traditional Chinese medicine. A total of 59.2 mg clemastanin B and 66.1 mg indigoticoside A with purities of 94.6% and 99.0% determined by high performance liquid chromatography (HPLC) were obtained in one-step elution from 250 mg crude extract, which contained clemastanin B 24.8% and indigoticoside A 28.4%, and the recoveries of clemastanin B and indigoticoside A were 90.3% and 92.2%, respectively. The chemical structure was identified by IR, MS, 1H NMR and 13C NMR.