Interleukin-10 (IL-10)-producing regulatory B (B_reg) cells suppress autoimmune disease, and increased numbers of B_reg cells prevent host defense to infection and promote tumor growth and metastasis by converting resting CD4⁺ T cells to regulatory T (T_reg) cells. The mechanisms mediating the induction and development of B_reg cells remain unclear. Here we show that IL-35 induces B_reg cells and promotes their conversion to a B_reg subset that produces IL-35 as well as IL-10. Treatment of mice with IL-35 conferred protection from experimental autoimmune uveitis (EAU), and mice lacking IL-35 (p35 knockout (KO) mice) or defective in IL-35 signaling (IL-12Rβ2 KO mice) produced less B_reg cells endogenously or after treatment with IL-35 and developed severe uveitis. Adoptive transfer of B_reg cells induced by recombinant IL-35 suppressed EAU when transferred to mice with established disease, inhibiting pathogenic T helper type 17 (T_H17) and T_H1 cells while promoting T_reg cell expansion. In B cells, IL-35 activates STAT1 and STAT3 through the IL-35 receptor comprising the IL-12Rβ2 and IL-27Rα subunits. As IL-35 also induced the conversion of human B cells into B_reg cells, these findings suggest that IL-35 may be used to induce autologous B_reg and IL-35⁺ B_reg cells and treat autoimmune and inflammatory disease.