Rosmarinic acid and rutin were successfully separated from Glechoma hederaceaL. using high-speed counter-current chromatography for the first time. Eleven milligrams of rosmarinic acid (chromatographic purity 97.2 %) and 10mg of rutin (chromatographic purity 98.1 %) were obtained from 100mg ethyl acetate extract and 100mg n -butanol extract of Glechoma hederacea L., respectively, with the separation procedure less than 2h. Their structures were characterized by UV, MS, and NMR. The established methods were simple, fast, and convenient, which can be applied to the preparation of reference substances of rosmarinic acid and rutin.