Conclusion. Injection of endotoxin into the middle ear causes production of macrophage migration inhibitory factor (MIF) in an experimental mouse model of otitis media with effusion (OME). Down-regulation of MIF may become a new approach for the management of OME. Objective. To determine the role of MIF in OME. Materials and methods. Mice were divided into two groups and their middle ears were injected with either endotoxin or phosphate-buffered saline (PBS). Mice were sacrificed at 6 h, 12 h, or 1, 3, 7, or 14 days after injection and concentrations of MIF, interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) in middle ear effusions were measured by enzyme-linked immunosorbent assay. Results. Concentrations of MIF in the endotoxin group at I day and 3 days were significantly higher than in the PBS control group. Concentrations of IL-1 beta in the endotoxin group at 6 h, 12 h, 1 day, and 3 days were significantly higher than in controls. Concentrations of TNF-alpha in the endotoxin group at I day and 3 days were significantly higher than in controls. Concentration of MIF in the endotoxin group was positively correlated with that of IL-1 beta and TNF-alpha.