Tim-1, a type I transmembrane glycoprotein, consists of an IgV domain and a mucin domain. The IgV domain is essential for binding Tim-1 to its ligands, but little is known about the role of the mucin domain, even though genetic association of TIM-1 with atopy/asthma has been linked to the length of mucin domain. We generated a Tim-1–mutant mouse (Tim-1 ^Δmucin ) in which the mucin domain was deleted genetically. The mutant mice showed a profound defect in IL-10 production from regulatory B cells (Bregs). Associated with the loss of IL-10 production in B cells, older Tim-1 ^Δmucin mice developed spontaneous autoimmunity associated with hyperactive T cells, with increased production of IFN-γ and elevated serum levels of Ig and autoantibodies. However, Tim-1 ^Δmucin mice did not develop frank systemic autoimmune disease unless they were crossed onto the Fas-mutant lpr mice on a C57BL/6 background. Tim-1 ^Δmucin lpr mice developed accelerated and fulminant systemic autoimmunity with accumulation of abnormal double-negative T cells and autoantibodies to a number of lupus-associated autoantigens. Thus, Tim-1 plays a critical role in maintaining suppressive Breg function, and our data also demonstrate an unexpected role of the Tim-1 mucin domain in regulating Breg function and maintaining self-tolerance.