The role of B cells in the development of CD4+ regulatory T cells has been emphasized recently. Our previous studies have demonstrated that the antigen-presenting splenic B cells converted naïve CD4+CD25- T cells into CD4+CD25+Foxp3- T cells without additional cytokines or chemicals with regulatory activity and that referred to as Treg-of-B cells. The present study further showed that Treg-of-B cells increased the IL-10-producing population, and the expression of c-Maf, inducible T-cell co-stimulator (ICOS) as well as cytotoxic T-lymphocyte-associated protein 4 (CTLA4) after repeated stimulation of B cells in a cell-cell contact-dependent manner. Long-term cultured Treg-of-B cells exerted IL-10 and CTLA4-mediated antigen-specific suppressive activity; moreover, the single antigen-specific Treg-of-B cells inhibited in a non-antigen-specific fashion. In conclusion, these results suggest that repeated stimulation of B cells induced IL-10-producing CD4+Foxp3- regulatory T cells in a contact-dependent manner and these Treg-of-B cells possess IL-10 and CTLA4-dependent suppressive function.