Three new triterpenoids, 3alpha,27-dihydroxylup-20(29)-en-28-oic acid methyl ester, 3alpha-acetoxy-27-hydroxylup-20(29)-en-28-oic acid methyl ester, and 3alpha-acetoxyolean-12-ene-27,28-dioic acid 28-methyl ester, were isolated from the roots of Peganum nigellastrum along with four known lupene-type triterpenoids. The structures of the new triterpenoids were determined by NMR spectroscopic means. The new triterpene, 3alpha, 27-dihydroxylup-20(29)-en-28-oic acid methyl ester is a DNA topoisomerase II inhibitor (IC(50) = 8.9 microM/mL).