High-speed counter-current chromatography (HSCCC) was applied to the isolation and purification of lutein from microalgae. Analytical HSCCC was used for the preliminary selection of a suitable solvent system composed of n-hexane-ethanol-water (4:3:1, v/v). Using the above solvent system, preparative HSCCC was successfully performed yielding lutein at 98% purity from 200 mg of the crude extract in a one-step separation.