Impaired regulatory B cell (Breg) responses are associated with several autoimmune diseases in humans; however, the role of Bregs in type 1 diabetes (T1D) remains unclear. We hypothesized that naturally occurring, interleukin-10 (IL-10)–producing Bregs maintain tolerance to islet autoantigens, and that hyperglycemic nonobese diabetic (NOD) mice and T1D patients lack these potent negative regulators. IgVH transcriptome analysis revealed that islet-infiltrating B cells in long-term normoglycemic (Lnglc) NOD, which are naturally protected from diabetes, are more antigen-experienced and possess more diverse B-cell receptor repertoires compared to those of hyperglycemic (Hglc) mice. Importantly, increased levels of Breg-promoting CD40+ B cells and IL-10–producing B cells were found within islets of Lnglc compared to Hglc NOD. Likewise, healthy individuals showed increased frequencies of both CD40+ and IL-10+ B cells compared to T1D patients. Rituximab-mediated B-cell depletion followed by adoptive transfer of B cells from Hglc mice induced hyperglycemia in Lnglc human CD20 transgenic NOD mouse models. Importantly, both murine and human IL-10+ B cells significantly abrogated T-cell–mediated responses to self- or islet-specific peptides ex vivo. Together, our data suggest that antigen-matured Bregs may maintain tolerance to islet autoantigens by selectively suppressing autoreactive T-cell responses, and that Hglc mice and individuals with T1D lack this population of Bregs.