Macrophage migration inhibitory factor (MIF) exerts either a protective or a deleterious role in the immune response to different pathogens. We analyzed herein the role of MIF in the host control of toxoplasmosis using MIF^-/- mice backcrossed to either the BALB/c or the C57BL/6 genetic backgrounds. Both, wild-type (WT) BALB/c and MIF^-/- BALB/c mice were susceptible to infection with highly virulent RH as well as moderately virulent ME49 strains of T. gondii. MIF^-/- mice, however, showed greater liver damage and more brain cysts, produced less proinflammatory cytokines, and succumbed significantly faster than WT mice. Bone marrow-derived dendritic cells (BMDCs) from MIF^-/- mice produced less interleukin-1, interleukin-12, and tumor necrosis factor- than WT BMDCs after stimulation with soluble Toxoplasma antigen (STAg). Similar observations were made in CD11c⁺ low-density cells isolated from the spleens of MIF^-/- mice challenged with STAg. MIF^-/- C57BL/6 mice succumbed to ME49 infection faster than their WT counterparts. C57BL/6 mice that succumbed to infection with the ME49 strain produced less MIF than resistant BALB/c mice similarly infected. Interestingly, an analysis of brains from patients with cerebral toxoplasmosis showed low levels of MIF expression. Together, these findings demonstrate that MIF plays a critical role in mediating host resistance against T. Gondii.—Flores, M., Saavedra, R., Bautista, R., Viedma, R., Tenorio, E. P., Leng, L., Sánchez, Y., Juárez, I., Satoskar, A. A., Shenoy, A. S., Terrazas, L. I., Bucala, R., Barbi J., Satoskar, A. R., Rodriguez-Sosa, M. Macrophage migration inhibitory factor (MIF) is critical for the host resistance against Toxoplasma gondii..