Phloretin is one of the main polyphenols found in apple tree bark. The separation and purification of phloretin from the crude phloretin extract of apple tree bark was achieved by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (2:2:1:2, v/v) on a preparative scale. The lower phase was used as the mobile phase in the head-to-tail elution mode. Double separations were performed with the same two-phase solvent system. 39.2 mg of phloretin were obtained from 767.3 mg of the crude phloretin extract, which contained 5.72% of phloretin. The purity of phloretin was 98.2%, and the recovery was 88.7%. All HSCCC fractions were analyzed by high-performance liquid chromatography and the chemical structure of target compound was identified by UV, IR, ESI-MS, H-1 NMR and C-13 NMR analysis. The optimum extracting conditions of phloretin from apple tree bark were also investigated by orthogonal test. (C) 2010 Elsevier B.V. All rights reserved.