Control of infection caused by Leishmania major requires the development of IFN-gamma+CD4+ lymphocytes for the induction of microbicidal activity in host macrophages. We recently reported on the inability of conventionally resistant C57BL/6 mice to successfully resolve infection by an isolate of L. major, despite a strong IFN-gamma response by the host. Susceptibility was caused by Ag-specific IL-10 from CD4+ cells that were also producing IFN-gamma. In the present studies, we have explored the role for IL-27 in the regulation of IL-10 from Th1 cells in leishmaniasis. Cytokine analysis of CD4+ cells in the lesions and draining lymph nodes of infected IL-27R-deficient (WSX-1(-/-)) mice revealed diminished IL-10 from IFN-gamma+ CD4+ cells, which was accompanied by a reduction in total IFN-gamma+CD4+ cells and an increase in IL-4. Despite the inhibition of IL-10 from CD4+ cells, no significant change in parasite numbers was observed, due both to the shift in the Th1/Th2 balance and to residual levels of IL-10. Strikingly, infected WSX-1(-/-) mice developed more severe lesions that were associated with the appearance of IL-17+ CD4+ cells, demonstrating a function for IL-27 in blocking the development of inappropriate Th17 cells during L. major infection. The results demonstrate the pleiotropic effects that IL-27 has on L. major-driven Th1, Th2, and Th17 development, and reinforce its function as a key regulatory cytokine that controls the balance between immunity and pathology.