Abstract: To investigate a suitable method for preparation of diosmin from Mentha × gracilis. The stems and leaves of Mentha × gracilis was extracted with 65% ethanol and purificated through AB-8 macroporous resin. A solvent system of ethyl acetate-n-butanol-water-acetic acid (3:1:3:0.005, V/V) was used for isolation and preparation by HSCCC (high speed counter-current chromatography). The upper phase was used as the stationary phase, while the lower one was used as mobile phase. The optimum purification conditions were as follow: isolation was performed at a flow-rate of 0.8 mL/min under rotator speed of 1600 r/min. Temperature was 25℃ and detection wavelength was 254 nm. Fraction II were collected according to HSCCC chromatogram and then detected by HPLC, which was diosmin with the purity of 95.2%.