NOD mice spontaneously develop anti-insulin autoantibodies and diabetes. A dominant peptide recognized by T-cell clones from NOD mice is insulin B-chain peptide B9-23. When administered subcutaneously to NOD mice, this peptide decreases the development of diabetes. In this study, we evaluated the autoantibody response to native insulin after administration of the B9-23 peptide. In NOD mice, administration of the B9-23 peptide in incomplete Freund’s adjuvant enhanced their insulin autoantibody response with a higher level and longer persistence. Induction of insulin autoantibodies with the B9-23 peptide was observed in non–diabetes-prone BALB/c mice and NOR mice within 2 weeks of administration, but this was not observed in C57BL/6 mice. A series of A-chain, other B-chain, and proinsulin peptides did not induce insulin autoantibodies. Induced anti-insulin autoantibodies could not be absorbed with the peptide alone but could be absorbed with native insulin. The B13-23 peptide (one of two identified epitopes within B9-23) when administered to BALB/c mice, induced autoantibodies, whereas peptide B9-16 did not. Induction of autoantibodies mapped to the major histocompatibility complex (MHC) rather than to the background genes. Both splenocytes with I-A^d/I-E^d or I-A^g7/I-E^null presented the B9-23 peptide to NOD islet-derived T-cell clones. Finally, administration of the B9-23 peptide to BALB/c mice, even without adjuvant, could induce insulin autoantibodies. Our results indicate that B-cell tolerance to intact insulin is readily broken with the presentation of the B9-23 insulin peptide, depending on the host’s specific MHC.