Triptolide (CAS 38748-32-2), a major active component of Tripterygium wilfordii Hook F, engages in multiple pharmacological activities. However, it interferes with the synthesis of sex steroid hormones. This was proven in the present study of male and female subjects with severe reproductive toxicities related to abnormal androgen or estrogen levels. The inhibitory action of triptolide on aromatase, a terminal enzyme responsible for the formation of estrogens from androgens, was determined in vitro to investigate its role in endocrine secretion. Triptolides were incubated with human placental microsomes for 20 min at concentrations of 10, 20, 30, and 40 nM. At 20 nM, triptolide significantly inhibited aromatase, as shown by [3H]2O assay, and its concentration at 50% inhibition (IC50) was determined as approximately 35 nM. Furthermore, the inhibitory effect of triptolide on aromatase was observed in human placental choriocarcinoma (JEG-3) cell lines. Treatment with 10 nM triptolide significantly inhibited the aromatase of JEG-3 cells and gave an IC50 value of approximately 17 nM. Under these concentrations, decreased levels of mRNA and protein expression of aromatase in JEG-3 cells were observed using Western blot analysis and quantitative real-time reverse transcription polymerase chain reaction assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test revealed that treatment with triptolide for 24 h under these concentrations did not trigger evident cell death. These findings characterize the molecular mechanisms of triptolide on the regulation of sex steroid hormones.