Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that is involved in the host defense against several pathogens. Here we used MIF^−/− mice to determine the role of endogenous MIF in the regulation of the host immune response against Trypanosoma cruzi infection. MIF^−/− mice displayed high levels of blood and tissue parasitemia, developed severe heart and skeletal muscle immunopathology, and succumbed to T. cruzi infection faster than MIF^+/+ mice. The enhanced susceptibility of MIF^−/− mice to T. cruzi was associated with reduced levels of proinflammatory cytokines, such as tumor necrosis factor alpha, interleukin-12 (IL-12), IL-18, gamma interferon (IFN-γ), and IL-1β, in their sera and reduced production of IL-12, IFN-γ, and IL-4 by spleen cells during the early phase of infection. At all time points, antigen-stimulated splenocytes from MIF^+/+ and MIF^−/− mice produced comparable levels of IL-10. MIF^−/− mice also produced significantly less Th1-associated antigen-specific immunoglobulin G2a (IgG2a) throughout the infection, but both groups produced comparable levels of Th2-associated IgG1. Lastly, inflamed hearts from T. cruzi-infected MIF^−/− mice expressed increased transcripts for IFN-γ, but fewer for IL-12 p35, IL-12 p40, IL-23, and inducible nitric oxide synthase, compared to MIF^+/+ mice. Taken together, our findings show that MIF plays a role in controlling acute T. cruzi infection.