Nitric oxide (NO) has emerged as a potent regulator useful in alleviating estrogen-deficiency bone loss. Osteoprotegerin (OPG) and receptor activator of NF-kB ligand (RANKL) play important roles in regulating osteoclastogenesis. While recent studies have reported NO donor attenuation of bone loss, the effect of NO donor on OPG and RANKL expression of osteogenic stromal cells and bone microenvironment in ovariectomized rats is not fully understood. Here, we showed that optimal NO donor treatment (2,2'-(hydroxynitrosohydrazino)bis-ethanamine; NOC-18, 15 µM) promoted OPG, but not RANKL levels in bone-marrow stromal cells from ovariectomized rats. NO donor augmentation of OPG synthesis was transcriptionally mediated. The stimulatory action of NO donor on OPG expression appeared to be regulated by tyrosine kinase-dependent activation of Cbfa1/Runx2 binding to the OPG promoter, because cell cultures pretreated with the tyrosine kinase inhibitor (herbimycin A), but not with the protein kinase A inhibitor (calphostain C) or the protein kinase C inhibitor ((Rp)-cAMP) significantly reduced NO-augmented Runx2 activation and OPG levels. Conditioned medium from NO donor-treated cells inhibited M-CSF and RANKL-induced osteoclast formation of M-CSF-dependent bone marrow marcophages. Neutralization with anti-OPG antibodies abolished the inhibitory effect of conditioned medium on osteoclastogenesis. Immunohistochemical observation also showed that NOC-18 increased OPG expression of osteochondral cells located at metaphyseal endosteum and calcified cartilage of proximal femurs in ovariectomized rats. These findings suggest that NO donor can be an alternative pharmacological strategy for regulating bone resorption.