This study is to investigate effects of genistein on rat femoral bone metabolic in vitro.  Rat femoral tissues was isolated and randomly divided into two groups including control group and genistein (1×10⁻⁵ mol·L⁻¹) group.  Determinations of alkaline phosphatase (ALP) activity, calcium content and osteoprotegerin (OPG), type I-collagen (Collagen-I), RANKL, Runx-2 and bone morphogenetic protein (BMP-2) mRNA expression were done by real-time PCR.  The results showed that 1×10⁻⁵ mol·L⁻¹ genistein could increase the activity of ALP and contents of Ca, regulate bone metabolism activity of OPG, RANKL, BMP-2, Collagen-I and Runx-2 mRNA expression level.  Genistein can significantly modulate bone metabolism related gene expression level of rat femoral tissue in vitro, and can increase calcium content and the activity of ALP.