Chagas disease was discovered more than a hundred years ago, but its pathogenesis is still not completely understood. Autoimmunity is one of the mechanisms shown to contribute to its pathogenesis, which may indicate an important participation of B lymphocytes. Patients with Chagas disease have shown increased percentage of B cells producing IL-10. However, there are no reports of the phenotypic markers of B cells producing IL-10 in patients with Chagas disease. For the first time in the literature, we evaluated the phenotypic profile of distinct markers of B cells from peripheral blood of noninfected individuals and patients with Chagas disease. Our results showed that patients with Chagas disease had a higher expression of CD21 and CD24 on the surface of CD19+ B cells, while CD43 and CD23 were expressed equally in all groups. Moreover, the expression of MHC-II (HLA-DR), CD80, CD86, caspase-3, granzyme B and intracellular IL-10 and TGF-β by CD19+ B cells was higher in patients with Chagas disease. The results of IL-10 production within CD19+ CD5+ CD1d+ B cells showed a higher percentage of this cytokine in patients with Chagas disease. Thus, our data bring a new knowledge about distinct markers of B cells in immune responses of Chagas disease.