Ultrahigh pressure extraction (UPE) was employed to extract podophyllotoxin and 4′-demethylpodophyllotoxin from Dysosma versipellis. The effects of extraction parameters including extraction solvents, pressure, time and solid/liquid ratio were investigated using a High Hydrostatic Pressure Processor. The optimal condition for UPE of the target compounds was 80% methanol, 200MPa of pressure, 1min of extraction time and 1:12 (g/mL) of solid/liquid ratio. Podophyllotoxin and 4′-demethylpodophyllotoxin in the crude extract were purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (10:10:8:12, v/v), and the fractions were analyzed by HPLC, ESI-MS and 1 H NMR. As a result, 73.7mg podophyllotoxin and 16.5mg 4′-demethylpodophyllotoxin with purities over 96% were obtained from 260mg crude sample in one-step separation.