Isoquercitrin and quercitrin were successfully isolated and purified from Hypericum japonicum Thunb.ex Murray by counter-current chromatography with a solvent system of n-hexane-ethyl acetate-methanol-water (1:7:1:7, v/v/v/v) in one step. From 100mg of the extract of Hypericum japonicum Thunb.ex Murray, 9.8mg of isoquercitrin and 12mg of quercitrin were obtained with the purities of 95.9% and 99.1%, respectively, as determined by HPLC. Their structures were identified by UV, MS, and NMR analysis. In this study, a rapid method for isolation and purification of the two major compounds from Hypericum japonicum Thunb.ex Murray crude extract was established.