Ergosterol　and　stigmasterol　are　the　most　common　phytosterols　in　the　traditional　Chinese　medicine.　They　are　two　major　sterol　compounds　in　Anoectochilus　roxburghii　(wall)　Lindl　(A.　roxburghii)　and　have　been　proved　to　have　many　important　biological　activities.　A　method　by　using　high-speed　counter-current　chromatography　(HSCCC)　has　been　successfully　developed　for　separation　and　purification　of　ergosterol　and　stigmasterol　in　A.　roxburghii　simultaneously　in　this　paper.　The　optimum　conditions　used　in　this　method　were　as　follows:　The　two-phase　solvent　system　consisted　of　n-hexane-ethylacetate-　butanol-methanol-water　(3.5:0.3:0.5:2.5:0.3,　v/v);　the　rotation　speed　was　900　rpm;　the　flow　rate　of　the　lower　phase　was　1.5　mL/min.　About　36.5　mg　of　ergosterol　and　43.6　mg　of　stigmasterol　were　obtained　from　100　g　of　A.　roxburghii.　The　purity　of　ergosterol　and　stigmasterol　was　examined　to　be　92.0　and　95.5%,　respectively,　by　using　HPLC.　The　chemical　structures　of　these　components　were　identified　by　UV　spectra,　FT-IR,　MS,　1H-NMR　and　13C-NMR.　The　results　demonstrated　that　high-speed　counter-current　chromatography　was　a　feasible　method　to　separate　and　purify　ergosterol　and　stigmasterol　from　the　herb.　This　separation　and　purification　method　was　more　effective　than　many　other　conventional　techniques.　Copyright　©　2011　WILEY-VCH　Verlag　GmbH　&　Co.　KGaA,　Weinheim.