INTRODUCTION: The main chemical constituents of Caesalpinia sappan are homoisoflavonoids. Conventional column chromatographic techniques used for isolation of this type of compounds are tedious, time-consuming and waste solvents. High-speed counter-current chromatography (HSCCC) could be a suitable alternative for the enrichment and purification of these target compounds from traditional Chinese medicine (TCM). OBJECTIVE: To establish a method to isolate four homoisoflavonoids in one-step separation from C. sappan by HSCCC. METHODOLOGY: The crude extract of C. sappan was fractionated by HSCCC using a two-phase solvent system consisting of chloroform-methanol-water (4:3:2, v/v/v). The separation conditions were: flow rate, 1.0 mL/min; revolution speed, 900 rpm; detection wavelength, 280 nm; separation temperature, 25 °C; sample size, 120 mg crude sample dissolved in a mixture of the upper and lower phases (10 mL each). The retention of the stationary phase was 83%. RESULTS: Five milligrams of 3'-deoxysappanol, 8 mg of 3-deoxysappanone B, 20 mg of 4-O-methylsappanol and 18 mg of brazilin were obtained in one-step separation from 120 mg of an ethyl acetate extracted fraction of C. sappan. Their purities were 99%, 97%, 90% and 85% by HPLC analysis. The mean recoveries of the four compounds were 83%, 86%, 93% and 85%, respectively. CONCLUSION: The study has shown that HSCCC is effective for the separation and enrichment of the target compounds at a large scale. Copyright © 2011 John Wiley & Sons, Ltd.