Leishmania infantum is the causative agent of zoonotic visceral leishmaniasis (ZVL), a disease frequently characterized by specific impairment of cell-mediated immune responses and uncontrolled parasitization. Regulatory T cells (Treg) have been shown to be involved in the direct induction of immunosuppression of effector immune response during chronic Leishmania infections. The present study aims to investigate the possible involvement of Treg cells during L. infantum infection. Results indicate that CD4(+)CD25(+) regulatory T cells are present in L. infantum-infected BALB/c mice and exhibit phenotypic and functional characteristics of Treg. The presence of high levels of Foxp3 gene expression and surface expression of alpha(E)beta(7) integrin (CD103) suggest a predisposition for Treg retention within sites of L. infantum infection, as is the case of the spleen and draining lymph nodes, consequently influencing local immune response. Th1 and Th2 effector immune responses seem inadequate, due to Treg expansion. Foxp3 expressing CD4(+)CD25(+) T cells are capable of producing TGF-beta and may contribute to immunosuppression and better control of parasite-mediated-immunopathology during infection. Surprisingly, IL-10 producing-CD4(+)CD25(-)Foxp3(-) T cells were also identified as an additional source of IL-10 and may represent a type 1 regulatory T (Tr1) cell subset that is being induced by L. infantum parasites. These findings suggest that distinct regulatory T cells develop in response to L. infantum and may play a possible role in promoting parasite persistence and the establishment of chronic infection.