Abstract: OBJECTIVE To observe whether puerarin promotes rat bone marrow mesenchymal cells osteogenesis differentiation through NO signaling pathway in vitro. METHODS The rat bone marrow mesenchymal cells were pretreated with nitric oxide synthase inhibitors(LNMA) 12 h, to observe the presence of puerarin on osteocalcin(OC), alkaline phosphatase(ALP) and calcification nodules staining, PCR detected the expression levels of osteogenic genes Runx-2, OSX, BMP-2, and Collagen-1 mRNA and determined NO and 3'-5'-Cycloguanosine monophosphoric acid(3'-5'-cyclic-GMP, cGMP) after different processing times. RESULTS After treatment of rat bone marrow mesenchymal cells with different concentrations of puerarin, the group of 1×10^-6 mol·L^-1significantly increased the activity of ALP in rat bone marrow mesenchymal cells and pre-treated with LNMA. After the rat bone marrow mesenchymal cells. The enhancement of ALP activity, OC content, calcification ability, and expression levels of Runx-2, OSX, BMP-2, and Collagen-1 mRNA in rat bone marrow mesenchymal cells were inhibited. It was further verified that puerarin could significantly increase NO and cGMP content, but the pretreatment with LNMA was inhibited. CONCLUSION puerarin can effectively promote the maturation and mineralization of rat bone marrow mesenchymal cells in vitro, and NO signaling pathway is required to participate.