To extract and separate Acanthoside-D contained in Acanthopanax Senticosus as a ginseng-like substance, the optimum operating conditions were experimentally determined in the analytical and preparative chromatography. Acanthoside-D was extracted from the powder of the trunk of Acanthopanax Senticosus by enthanol, and the resulting solution was partitioned with n-hexane. The mobile phase composition and injection volume by analytical chromatography were water/acetonitrile/methanol=80/14/6 vol% and 20 μl, respectively while those by recycle chromatography were 70/15/15 vol% and 2 ml. By recycling the sample 4 times, Acanthoside-D with 93% purity was collected.