Salaspermic acid [1], an inhibitor of HIV reverse transcriptase and HIV replication in H9 lymphocyte cells, was isolated from the roots of Tripterygium wilfordii for the first time. The structure of 1 derived from spectral data was established unequivocally by an X-ray analysis of crystals of the monohydrate. A structure-activity correlation of 1 with ten related compounds indicated that the acetal linkage in ring A and the carboxyl group in ring E of 1 may be required for the anti-HIV activity.