IL-10-competent regulatory B cells (B10 cells) are a functionally unique and numerically rare (1-3%) subset of mouse spleen B cells that have the capacity to negatively regulate adaptive, innate and humoral immunity, as well as cellular immune responses in mouse models of inflammation and autoimmunity. B10 cell development and regulation of immune responses requires appropriate B cell antigen receptor (BCR) signals and the expression of a diverse BCR repertoire, implying antigen-specific interactions with T cells. In fact, B10 cell maturation into functional IL-10-secreting effector cells that inhibit in vivo autoimmune disease requires IL-21 and CD40-dependent cognate interactions with T cells. However, the exact antigens that drive B10 cell expansion and IL-10 secretion in vivo are unknown. Using IL-10 reporter mice and a mouse model of contact hypersensitivity (CHS), here we show that ≥60% of spleen B10 effector cells actively secreting IL-10 under homeostatic conditions (0.01 to 0.1% of spleen B cells) are driven to do so by self-antigens. Furthermore, during active inflammation, B10 cells expand in frequency and regulate immune responses in an antigen-specific manner. The ex vivo provision of CD40 and IL-21 receptor signals can also drive the development and 30,000-fold expansion of B10 cells that dramatically inhibit antigen-specific disease symptoms when transferred into mice with CHS or established autoimmune disease. Thereby, the ex vivo expansion and reinfusion of autologous B10 cells may provide a novel and effective in vivo treatment that facilitates tolerance in patients with autoimmune diseases that are resistant to current therapies.