The hyperosmotic stimulus is regarded as a mechanical factor for bone remodeling. However, whether the hyperosmotic stimulus affects 1Ձ, 25-dihydroxyvitamin D3 (1Ձ,25(OH)2D3)-induced osteoclastogenesis is not clear. In the present study, the effect of the hyperosmotic stimulus on 1Ձ,25(OH)2D3- induced osteoclastogenesis was investigated in an osteoblast-preosteoclast co-culture system. Serial doses of sucrose were applied as a mechanical force. These hyperosmotic stimuli significantly evoked a reduced number of 1Ձ,25(OH)2D3-induced tartrate-resistant acid phosphatase-positive multinucleated cells and 1Ձ,25(OH)2D3-induced bone-resorbing pit area in a co-culture system. In osteoblastic cells, receptor activator of nuclear factor ՊB ligand (RANKL) and Runx2 expressions were down-regulated in response to 1Ձ,25(OH)2D3. Knockdown of Runx2 inhibited 1Ձ,25(OH)2D3-induced RANKL expression in osteoblastic cells. Finally, the hyperosmotic stimulus induced the overexpression of TonEBP in osteoblastic cells. These results suggest that hyperosmolarity leads to the down-regulation of 1Ձ,25(OH)2D3- induced osteoclastogenesis, suppressing Runx2 and RANKL expression due to the TonEBP overexpression in osteoblastic cells.