Supercritical fluid extraction (SFE) of aurentiamide acetate from Patrinia villosa Juss was performed. The optimization of parameters was carried out using an analytical-scale supercritical fluid extraction (SFE) system. Then the extraction was scaled up by 100 times using a preparative SFE system under the optimized conditions of 55 degrees C, 35 MPa and modified CO2 with 10% methanol. Then, the crude extract I obtained by SFE was chromatographed on silica gel and the solvent system composed of petroleum ether-ethyl acetate (5:1, v/v) was used to produce the crude extract II, which was further isolated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:1.2:1.2:1, v/v/v/v). One hundred fifty-five milligrams of aurentiamide acetate was obtained from 400 mg crude extract II (contained 42% target) with a purity of 99.3% determined by HPLC and 92.3% recovery in one-step elution, and identification was performed by UV, MS, 1H NMR and 13C NMR. As far as we know, this is the first report of discovering aurentiamide acetate from the plant of Patrinia genius.