A number of studies have suggested that B cell mediated-regulation contributes to the establishment of immunological tolerance. However, the precise mechanisms by which regulatory B cells establish and maintain tolerance in humans remain to be determined. The objective of the current study is to understand the cellular and molecular bases of B-cell regulatory functions in humans. To describe the mechanisms regulating the functional plasticity of regulatory B cells, we used an in vitro co-culture model based on autologous mixed lymphocyte cultures involving freshly isolated B and T cells. The results show that activated B cells regulate T cell proliferation through producing transforming growth factor (TGF)-β and indoleamine 2,3-dioxygenase (IDO). The production of TGF-β and IDO leads to the induction of not only “natural” regulatory T cells but also of TGF-β-producing CD4⁺ T cells and IL-10-producing regulatory T cells. Furthermore, we evidenced for the first time that CTLA-4 induces B-cells to produce IDO and to become effective induced regulatory B cells (iBregs). This study emphasizes a novel regulatory axis and open news insights in how to manage regulatory B cell functions in autoimmunity.