ETHNOPHARMACOLOGICAL RELEVANCE: Epimedium brevicornum Maxim (E. brevicornum) has long been used for the treatment of osteoporosis in China and other Asian countries. However, the mechanism behind the antiosteoporotic activity of this medicinal plant is not fully understood. AIM OF THE STUDY: The present study was designed to investigate the effects of 5 widely used antiosteoporotic medicinal plants (E. brevicornum, Cuscuta chinensis, Rhizoma drynariae, Polygonum multiflorum, and Ligustrum lucidum) on the production of estrogen and identify the bioactive compounds responsible for the estrogen biosynthesis-promoting effect. MATERIALS AND METHODS: Human ovarian granulosa-like KGN cells were used to evaluate estrogen biosynthesis, and the production of 17β-estradiol was quantified by magnetic particle-based enzyme-linked immunosorbent assay (ELISA) kit. Further, the mRNA expression of aromatase was determined by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR), and the protein expression of aromatase was detected by western blotting. The activity of alkaline phosphatase (ALP) in rat osteoblastic UMR-106 cells was measured using p-nitrophenyl sodium phosphate assay. RESULTS: Among the 5 antiosteoporotic medicinal plants, the extract of E. brevicornum was found to significantly promote estrogen biosynthesis in KGN cells. Icariin, the major compound in E. brevicornum, was identified to be the active compound for the estrogen biosynthesis-promoting effect. Icariin promoted estrogen biosynthesis in KGN cells in a concentration- and time-dependant manner and enhanced the mRNA and protein expressions of aromatase, which is the only enzyme for the conversion of androgens to estrogens in vertebrates. Further study showed that icariin also promoted estrogen biosynthesis and ALP activity in osteoblastic UMR-106 cells. CONCLUSIONS: These results show that the promotion of estrogen biosynthesis is a novel effect of E. brevicornum, and icariin could be utilized for the prevention and treatment of osteoporosis.