The serum protein prothrombin (PT) is proteolytically converted to thrombin during the coagulation cascade by the cell-associated prothrombinase complex. In vitro, RANKL-differentiated osteoclasts express tissue factor and coagulation factor Xa, which convert PT to thrombin (Karlstrom et al. Biochem Biophys Res Commun 394:593–599, 2010). The present study investigated the localization of PT in bone as well as the expression of PT mRNA in bone and osteoclasts. Herein, immunoblot analysis detected PT and smaller proteolytically cleaved fragments with sizes consistent with the action of prothrombinase in a protein fraction extracted with guanidine-HCl EDTA from mouse tibia. Light microscopic and ultrastructural immunohistochemistry demonstrated the presence of PT in the newly formed bone matrix of the metaphysis. Furthermore, fluorescent immunohistochemistry on metaphyseal trabecular bone showed that PT colocalized with MMP-9-expressing subepiphyseal osteoclasts, whereas cathepsin K-expressing osteoclasts were closely associated with PT of the bone matrix. RT-qPCR analysis revealed that PT mRNA was detected in tibia. Expression of PT mRNA in the tibia was 0.2% of the level in the liver. In addition, PT mRNA expression was increased by RANKL-induced differentiation of bone marrow macrophages to osteoclasts. The results demonstrate that PT is synthesized and proteolytically processed in bone. Furthermore, PT is present mainly in the newly formed bone matrix of the metaphyseal trabecular bone compartment in close association to osteoclasts. In addition, MMP-9-positive osteoclasts contain PT, and PT expression is increased during osteoclastogenesis.