Four hitherto unknown triterpenoids, including one euphane, namely wilfordeuphone (1), and three friedelanes, namely wilforic acids G − I (2–4), were isolated from the ethanol extract of the roots of Tripterygium wilfordii. The absolute configuration of 1 was established by single crystal X-ray diffraction. In addition, this study is the first to report euphane-type triterpenoid in the genus Tripterygium. Compound 1 exhibited the moderate cytotoxic activity when evaluated against five human cancer cell lines (HL-60, A-549, SMMC-7721, MCF-7, and SW-480), with IC50 values ranging from 14.0–26.7 μM.