The intracellular protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas' disease. We have previously characterized a T. cruzi virulence factor named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin protein family. Single mutant parasite clones (Tc52(+/-)) exhibiting low virulence in vitro and in vivo were obtained by targeted Tc52 gene replacement. In this report, we have extended our study to analyze the immune response and the disease phenotype in Tc52(+/-)-infected BALB/c mice, during the acute and chronic phases of the disease. Significantly lower parasitemia were found in Tc52(+/-)-infected mice, as compared to wild-type parasite (WT)-infected ones. However, the expansion of all classes of lymphocytes and macrophages was similar for both clones. Furthermore, except for IgG2b levels which were higher in the case of WT-infected mice, all classes of Ig presented no significant difference for WT and Tc52(+/-)-infected animals. Interestingly, a lack of suppression of IL-2 production and of T-cell proliferation inhibition was observed in the case of spleen cells from Tc52(+/-)-infected mice. Finally, the pattern of inflammation process was different and characterized as diffused in the case of Tc52(+/-)-infected mice, or presenting numerous foci in the case of WT-infected mice. Localization of the Tc52 protein in tissue sections and infected heart cell primary cultures by immunofluorescence and immunogold labeling, respectively, revealed the presence of Tc52 at the amastigote surface and associated to aggregates within host cell vesicles. Taken together, these results reinforce the notion of Tc52 being a virulence factor playing a role in the phenotype of the immune response associated to the infection and on the course of the disease.