Tolerance to African trypanosomes requires the production of IFN-gamma in the early stage of infection that triggers the development of classically activated macrophages controlling parasite growth. However, once the first peak of parasitemia has been controlled, down-regulation of the type 1 immune response has been described. In this study, we have evaluated whether regulatory T cells (Tregs) contribute to the limitation of the immune response occurring during Trypanosoma congolense infection and hereby influence the outcome of the disease in trypanotolerant C57BL/6 host. Our data show that Foxp3+ Tregs originating from the naturally occurring Treg pool expanded in the spleen and the liver of infected mice. These cells produced IL-10 and limited the production of IFN-gamma by CD4+ and CD8+ effector T cells. Tregs also down-regulated classical activation of macrophages resulting in reduced TNF-alpha production. The Treg-mediated suppression of the type 1 inflammatory immune response did not hamper parasite clearance, but was beneficial for the host survival by limiting the tissue damages, including liver injury. Collectively, these data suggest a cardinal role for naturally occurring Tregs in the development of a trypanotolerant phenotype during African trypanosomiasis.