Acanthoside-D from Acanthopanax Senticosus was extracted using carbon dioxide at supercritical state and the additive of water, and analyzed by reversed-phase high performance liquid chromatography (RP-HPLC). The chromatographic column was packed with Lichrospher 100RP-18 (15 μm) and the analysis was performed on the isocratic condition. The yield of Acanthoside-D in supercritical carbon dioxide with pressures, temperatures, and amounts of additive was investigated. In the experimental run without water additives, the yield of Acanthoside-D was increased with higher pressures. But the residual phase of supercritical extraction contained the larger amount of Acanthoside-D at the water additives than no additives. The sample was purified by solvent extraction and open tubular chromatography, and further isolated by RP-HPLC.