The 80% aqueous acetone extract of Zedoariae Rhizoma was found to show a protective effect against D-galactosamine (D-GalN)/lipopolysaccharide-induced acute liver injury in mice. To clarify the active compounds, the principal constituents were examined and 11 sesquiterpenes (furanodiene, curdione, neocurdrione, dehydrocurdione, germacrone, 13-hydroxygermacrone, curcumenol, isocurcumenol, aerugidiol, zedoarondiol, and curcumenone) and a diarylheptanoid (curcumin) were found to inhibit the increase in serum aspartate aminotransaminase and alanine aminotransaminase at a dose of 50 mg/kg p.o. in agreement with the previous in vitro studies, except for dehydrocurdione, aerugidiol, and zedoarondiol. In particular, curdione, neocurdione, curcumenol, and isocurcumenol potently inhibited the increase at a dose of 12.5 mg/kg p.o. Furthermore, the eight sesquiterpenes, furanodiene, curdione, neocurdione, dehydrocurdione, germacrone, 13-hydroxygermacrone, curcumenol, and curcumenone, also showed a protective effect against D-GalN/tumor necrosis factor-α-induced liver injury in mice at a dose of 50 mg/kg p.o.