Enrichment and separation of alkaloids from a chloroform extract of Sinomenium acutum has been successfully performed for the first time using pH-zone-refining counter-current chromatography. The two-phase solvent system used for enrichment was composed of Methyl tert-butyl ether (MtBE)-acetonitrile (CH3CN)-water (4:1:5, v/v), where 10mM triethylamine (TEA) was added to the upper organic stationary phase as a retainer and 10mM hydrochloric acid (HCl) to the aqueous mobile phase as an eluter, which could enrich the alkaloids from the crude extract well. For the preparative separation, the solvent system consisted of MtBE-CH3CN-water (4:0.5:5, v/v) with 10mM TEA in organic stationary phase and 5mM HCl in the aqueous mobile phase, which could separate and purify the enriched crude alkaloids successfully. 0.82g of crude alkaloids was enriched from 1.60g of chloroform extract in the first step separation. From the enriched crude alkaloids, 376mg of sinomenine and 85mg of acutumine were obtained in the second step separation with the purity of 98.1% and 98.7%, respectively. The chemical structures of the isolated compounds were identified by UV, ESI-MS and 1H NMR.