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Abstract: Alkaloids in Gelsemium elegans possess a variety of therapeutic properties, including tumor suppression, analgesic and anti-inflammatory effects. In China, G. elegans has been used for centuries to treat a variety of medical conditions, including chronic pain and skin ulcer. Methods currently used to separate the active components of G. elegans are time-consuming and have low recovery. In the present study, we used pH-zone-refining counter-current chromatography to separate major alkaloids from a crude extract of G. elegans. The two-phase solvent system was methyl tert-butyl ether (MtBE)/acetonitrile/water (3:1.5:4, v/v). Triethylamine (20mM) was added to the upper organic stationary phase as a retainer. Hydrochloric acid (10mM) was added to the lower aqueous phase as an eluter. From 1.5g of crude extract, we obtained 312mg gelsemine, 420mg koumine and 195mg gelsevirine, with purities at 94.8%, 95.9% and 96.7%, respectively, which were determined by HPLC at 256nm. The chemical identity of the isolated compounds was verified by electrospray ionization-mass spectrometry (ESI-MS), 1H NMR and 13C NMR. These results demonstrated that pH-zone-refining counter-current chromatography is an effective method to separate and purify major alkaloids from G. elegans. [Copyright &y& Elsevier]