An efficient separation method of using high-speed counter-current chromatography was successfully established to directly purify cytotoxic transformed products of cinobufagin by Cordyceps militaris. The two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (4:6:3:4, v/v) was used in high-speed counter-current chromatography. A total of 9 mg of 4beta,12alpha-dihydroxyl-cinobufagin (1), 15 mg of 12beta-hydroxyl-cinobufagin (2), 8 mg of 5beta-hydroxyl-cinobufagin (3), 12 mg of deacetylcinobufagin (4) and 6 mg of 3-keto-cinobufagin (5) were obtained in a one-step separation from 400 mg of the crude extract with purity of 98.7, 97.2, 90.6, 99.1 and 99.4%, respectively, as determined by HPLC. Their chemical structures were identified on the basis of (1)H-NMR and (13)C-NMR technology. All products (1-5) showed the potent activities against human carcinoma cervicis (Hela) and malignant melanoma (A375) cells in vitro.