High-speed counter-current chromatography (HSCCC) with a solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v/v) was used to isolate and separate two C-glycosylflavones from Patrinia villosa Juss, a traditional Chinese medicine. The separation produced 42.9 mg isovitexin and 20.1 mg isoorientin with purities of 99.3% and 98.5%, respectively as determined by high-performance liquid chromatography (HPLC) in one step elution from 250 mg crude extract, and identification was performed by MS, 1H NMR and 13C NMR. It is the first report of discovering isovitexin and isoorientin from the plant of Patrinia genus.