1α， 25-dihydroxyvitamin D3 (1α， 25(OH)2D3) acts on the osteoblasts to enhance the expressions of receptor activator of nuclear factor κB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) and induce the formation of osteoclasts. However， the mechanism in osteoblasts by which 1α， 25(OH)2D3 promotes osteoclastogenesis has not yet been completely understood. This study aimed to select the first generation of murine osteoblasts to explore the underlying mechanism of 1α， 25(OH)2D3-induced osteoclastic formation from bone marrow mononuclear cells (BMMNCs). We discovered the activation of osteoblastic NF-κB pathway under 10(-8) mol/L 1α， 25(OH)2D3 treatment， as evidenced by the transfer of NF-κB p65 from cytoplasm to nuclei. Then， the NF-κB p65-siRNA was designed， constructed， and transfected into osteoblastic cells. Immunofluorescence assay confirmed the successfully silenced NF-κB p65 gene in osteoblasts. In the co-culture system of osteoblasts and BMMNCs with 1α， 25(OH)2D3 added， the multinucleated osteoclast-like cells containing 2-3 nuclei were observed in BMMNCs co-cultured with non-transfection osteoblasts， conversely， silencing osteoblastic NF-κB p65 resulted in failed differentiation of BMMNCs along with substantial vacuolar degeneration in cytoplasm. In addition， the expressions of RANKL and M-CSF were notably decreased in NF-κB p65-silenced osteoblasts. Taken together， our data indicated that osteoblastic NF-κB pathway was involved in 1α， 25(OH)2D3-induced osteoclast-like cells formation from BMMNCs through regulating the expression of RANKL and M-CSF. Therefore， our findings further identified the mechanism of 1α， 25(OH)2D3-induced osteoclastogenesis on the basis of prior studies.