An efficient method using recycling high speed counter-current chromatography (HSCCC) with hydroxy propyl-beta-cyclodextrin (HP-beta-CD) as chiral selector (CS) is developed for the separation of ketoconazole enantiomers. The separation process was performed with a two-phase solvent system composed of n-hexane/acetic acid isobutyl ester/0.1 mol L-1 phosphate buffer solution with pH = 8.5 containing 0.1 mol L-1 HP-beta-CD (2:3:5, v/v/v). Important factors that influence the separation efficiency, including organic solvent system, temperature, the pH value of the aqueous phase as well as type and concentration of chiral selector were investigated experimentally. Influence of the pH value and concentration of chiral selector was modeled and the conditions were further optimized by the model. Pure enantiomers with a purity >99% were obtained by separating 20 mg racemate via HSCCC. The recovery rates ranged from 85% to 89%. (C) 2016 Elsevier B.V. All rights reserved.