Control of visceral leishmaniasis (VL) depends on pro-inflammatory Th1 cells that activate infected tissue macrophages to kill resident intracellular parasites. However, pro-inflammatory cytokines produced by Th1 cells can damage tissues, and require tight regulation. Th1 cell IL-10 production is an important cell-autologous mechanism to prevent such damage. However, IL-10-producing Th1 (type I regulatory; Tr1) cells can also delay control of parasites and the generation of immunity following drug treatment or vaccination. To identify molecules to target to alter the balance between Th1 and Tr1 cells for improved anti-parasitic immunity, we compared the molecular and phenotypic profiles of Th1 and Tr1 cells in experimental VL caused by Leishmania donovani infection of C57BL/6 mice. We also identified a shared Tr1 cell protozoan signature by comparing the transcriptional profiles of Tr1 cells from mice with experimental VL and malaria. We identified LAG3 as an important co-inhibitory receptor in VL patients and experimental VL, and reveal tissue-specific heterogeneity of co-inhibitory receptor expression by Tr1 cells. We also discovered a role for the transcription factor Pbx1 in suppressing CD4+ T cell cytokine production. This work provides insights into the development and function of CD4+ T cells during protozoan parasitic infections and identifies key immunoregulatory molecules.