Ginkgolic acids (GAs), bioactive compounds found in Ginkgo biloba L., have received much attention in pharmacological research. Separation of individual GAs from their mixtures is, however, difficult. Herein, an efficient method for the preparative separation of five major GAs from the sarcotesta of Ginkgo biloba L. was developed using beta-cyclodextrin (beta-CD) clathration coupled with pH-zone-refining countercurrent chromatography (PZRCCC) and recycling CCC. Clathration by beta-CD was employed to enhance the hydrophilicity of the GAs. Separation of beta-CD-GA clathrates was achieved by PZRCCC using a two-phase solvent system composed of n-heptane-ethyl acetate- methanol-water (2:1:1.5:1, v/v). The upper phase was acidified with 40 mM HCl (stationary phase), and the lower phase was made alkaline with 5 mM triethylamine. Three GAs were obtained with purities over 98% together with two mixtures. The mixtures were separated in recycling CCC mode using n-heptane-ethyl acetate-methanol-acetic acid (5:4:1:1, v/v) as the solvent system. Five GAs, including C13:0, C15:0, C15:1, C17:1, and C17:2, were successfully separated with purities over 98%, and their structures were confirmed by comparison with a mixture of GA standard samples coupled with ESI-MS data.