Locally produced osteoclastogenic factor RANKL plays a critical role in the development of bone resorption in periradicular periodontitis. However, because RANKL is also required for healthy bone remodeling, it is plausible that a costimulatory molecule that upregulates RANKL production in inflammatory periradicular periodontitis may be involved in the pathogenic bone loss processes. We hypothesized that macrophage migration inhibitory factor (MIF) would play a role in upregulating the RANKL-mediated osteoclastogenesis in the periradicular lesion. In response to pulp exposure, the bone loss and level of MIF mRNA increased in the periradicular periodontitis, which peaked at 14 d, in conjunction with the upregulated expressions of mRNAs for RANKL, proinflammatory cytokines (TNF-α, IL-6, and IL-1β), chemokines (MCP-1 and SDF-1), and MIF's cognate receptors CXCR4 and CD74. Furthermore, expressions of those mRNAs were found significantly higher in wild-type mice compared with that of MIF2/2 mice. In contrast, bacterial LPS elicited the production of MIF from ligament fibroblasts in vitro, which, in turn, enhanced their productions of RANKL and TNF-a. rMIF significantly upregulated the number of TRAP+ osteoclasts in vitro. Finally, periapical bone loss induced in wild-type mice were significantly diminished in MIF -/- mice. Altogether, the current study demonstrated that MIF appeared to function as a key costimulatory molecule to upregulate RANKL-mediated osteoclastogenesis, leading to the pathogenically augmented bone resorption in periradicular lesions. These data also suggest that the approach to neutralize MIF activity may lead to the development of a therapeutic regimen for the prevention of pathogenic bone loss in periradicular periodontitis.