Glycyrrhiza uralensis (licorice) is one of the most frequently prescribed ingredients in Oriental medicine, and licorice extract has been shown to exert anti-carcinogenic effects. However, its use as a cancer chemopreventive agent is rather limited, due to the fact that its principal component, glycyrrhizin, is known to induce hypertension. This study determined the effects of a hexane/ethanol extract of G. uralensis (HEGU), which contains undetectable amounts of glycyrrhizin, on the apoptosis of androgen-insensitive DU145 cells. HEGU induced apoptosis and increased the levels of cleaved caspase-9, caspase-7, caspase-3 and poly (ADP-ribose) polymerase (PARP). HEGU also induced mitochondrial membrane depolarization and cytochrome c release to the cytosol. HEGU increased the levels of Fas, death receptor 4 (DR4), cleaved caspase-8, Mcl-1S, and truncated Bid proteins. A caspase-8 inhibitor suppressed HEGU-induced apoptosis. An active fraction of HEGU was separated via column chromatography and the structure of the active compound isoangustone A was identified via 1H-NMR and 13C-NMR. Isoangustone A increased apoptotic cells, the cleavage of PARP and caspases, and the levels of DR4 and Mcl-1S. Transfection with DR4 small interfering RNA attenuated HEGU- and isoangustone A-induced apoptosis. These results demonstrate that the activation of DR4 contributes to HEGU- and isoangustone A-induced apoptosis of DU145 cells.