, a member of the tight junction family of proteins, is a negative regulator of RANKL-induced osteoclast differentiation and bone resorption (BR) in vivo. Since estrogen deficiency decreases bone mass in part by a RANKL-mediated increase in BR, we evaluated whether estrogen regulates Cldn-18 expression in bone. We found that Cldn-18 expression was reduced in the bones of estrogen deficient mice, whereas it was increased by estrogen treatment in osteoblasts and osteoclasts in vitro. We next evaluated the role of Cldn-18 in mediating estrogen-induced bone loss. Cldn-18 knockout (KO) and littermate wild-type (WT) mice were ovariectomized (OVX) or sham operated at 6 wk of age, and the skeletal phenotype was evaluated at 14 wk of age. PIXImus revealed that total body, femur, and lumbar BMD were reduced 8 -13% (P Ͻ 0.05) after 8 wk of OVX compared with sham in WT mice. As expected, total body, femur, and lumbar BMD were reduced 14 -21% (P Ͻ 0.05) in Cldn-18 KO sham mice compared with sham WT mice. However, ovariectomy failed to induce significant changes in BMD of total body, femur, or vertebra in the Cldn-18 KO mice. CT analysis of the distal femur revealed that trabecular (Tb) bone volume was decreased 50% in the OVX WT mice compared with sham that was caused by a 26% decrease in Tb number and a 30% increase in Tb separation (all P Ͻ 0.05). By contrast, none of the Tb parameters were significantly different in OVX Cldn-18 KO mice compared with sham KO mice. Histomorphometric analyses at the Tb site revealed that neither osteoclast surface nor osteoclast perimeter was increased significantly as a consequence of OVX in either genotype at the time point examined. Based on our findings, we conclude that the estrogen effects on osteoclasts may in part be mediated via regulation of Cldn-18 signaling.claudins; estrogen; bone resorption; bone density; osteoblasts; gene expression ESTROGEN DEFICIENCY IN MENOPAUSAL WOMEN is a serious health issue due to increased bone turnover, resulting in osteoporosis and increased fracture risk consequent to deterioration of microarchitectural bone structure (32). It is now recognized that estrogen prevents bone loss via multiple and complex effects on bone marrow and bone cells that result in decreased osteoclastogenesis, increased osteoclast apoptosis, and decreased capacity of mature osteoclasts to resorb bone (20). The canonical mechanism for osteoclast differentiation depends on activation of receptor activator of nuclear factor-B (RANK) by its ligand RANKL and tyrosine kinase receptor fms by macrophage colony-stimulating factor. Estrogen probably affects osteoclast differentiation in part indirectly through its modulation of osteoblast RANKL and osteoprotegerin expression (46) and directly by decreasing the responsiveness of osteoclast precursors to the RANKL (36). There is also evidence that estrogen blocks the production of the pro-osteoclastogenic cytokines IL-1, IL-6, and TNF (1, 18, 29). It has also been suggested that estrogen modulates osteoclast apoptosis and osteoclast acti...