Purpose: The balance between the activities of bone-forming osteoblasts and bone-resorbing osteoclasts (OCs) is important for maintaining bone homeostasis. Excessive activation of OCs causes bone-destruction diseases. Our previous studies showed that hypoxic condition promoted osteoclast formation. However, the precise mechanism of osteoclast formation under hypoxia has been unclear. In the present study, we investigated the role of inducible nitric oxide synthase (iNOS) on osteoclast differentiation under hypoxia. Method: Bone marrow cells obtained from mice were stimulated with receptor activator of NF-kappa B ligand (RANKL) and macrophage colony stimulating factor (M-CSF) to induce osteoclast differentiation. Bone marrow cells were cultured with iNOS inhibitor or NO (nitric oxide) donor under normoxia (O₂ 20%) or hypoxia (O₂ 5%). Results and Discussion: The number of osteoclasts was increased in the culture under hypoxia compared with that in the culture under normoxia. The gene and protein expression of iNOS increased in the culture under hypoxia. The addition of iNOS inhibitor in hypoxic culture reduced the number of osteoclasts. Addition of NO donors in normoxic culture, the number of osteoclasts were increased. These results suggested that hypoxic condition could promoted osteoclast differentiation via iNOS pathway.