Genistein, a soybean isoflavone, has estrogen-like activity in mammals, including the prevention of bone loss. However, whether its mechanism of action on bone turnover is distinct from that of estrogen or raloxifene is unknown. Although genistein has been reported to bind both estrogen receptor (ER) isoforms (α and β), little is known concerning differential activation of gene expression via these ER isoforms. To examine this question, comparison of the responses of normal fetal osteoblast (hFOB) cells stably expressing either ERα (hFOB/ERα9) or ERβ (hFOB/ERβ6), to treatment with genistein, 17β-estradiol (E2) or raloxifene were conducted. In hFOB/ERα9 cells, both genistein and E2 increased the endogenous gene expression of the progesterone receptor (PR), the proteoglycan versican, and alkaline phosphatase (AP), but inhibited osteopontin (OP) gene expression and interleukin-6 (IL-6) protein levels. Raloxifene had no effect on these bone markers. Genistein, but not raloxifene, also mimicked E2 action in the hFOB/ERβ6 cells increasing PR gene expression and inhibiting IL-6 production. To determine whether the gene regulatory actions of genistein in human osteoblast cells occur at the level of transcription, its action on the transcriptional activity of a PR-A promoter-reporter construct was assessed. Both genistein and E2 were found to stimulate the PR promoter in the hFOB cell line when transiently co-transfected with either ERα or ERβ. Whereas hFOB cell proliferation was unaffected by E2, raloxifene or genistein at low concentrations, higher concentrations of genistein, displayed significant inhibition. Together, these findings demonstrate that genistein behaves as a weak E2 agonist in osteoblasts and can utilize both ERα and ERβ. © 2003 Wiley-Liss, Inc.