Mineral trioxide aggregate (MTA) is a therapeutic, endodontic repair material that is reported to exhibit calcified tissue-conductive activity. The aim of this study was to investigate whether MTA may prevent osteoclast differentiation in vitro. MTA solution, but not other commonly used retrofilling materials, such as Dycal, Super-EBA, or intermediate restorative material (IRM) solution, dose-dependently inhibited osteoclastogenesis in cocultures of mouse bone marrow cells (BMCs) with primary osteoblast cells (POBs) induced by 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. Exogenous CaCl2 medium supplementation did not inhibit osteoclastogenesis in cocultures. Furthermore, MTA solution did not affect receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis, suggesting that POBs are targets of MTA. MTA solution suppressed the 1α,25(OH)2D3-induced reduction of osteoprotegerin (OPG) mRNA and protein production without changing RANKL expression in POBs. Consistent with this result, MTA solution did not inhibit osteoclastogenesis in cocultures of BMCs and POBs from OPG-deficient mice. Therefore, the maintenance of OPG expression in POBs appears to be critical for the inhibitory effect of MTA solution on osteoclast differentiation. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.