The cholera toxin B subunit (CTB) has been used as adjuvant to improve oral vaccine delivery in type 1 diabetes. The effect of CTB/peptide formulations on Ag-specific CD4+ T cells has remained largely unexplored. Here, using tetramer analysis, we investigated how oral delivery of CTB fused to two CD4+ T-cell epitopes, the BDC-2.5 T-cell 2.5mi mimotope and glutamic acid decarboxylase (GAD) 286–300, affected diabetogenic CD4+ T cells in nonobese diabetic (NOD) mice. When administered i.p., CTB-2.5mi activated 2.5mi+ T cells and following intragastric delivery generated Ag-specific Foxp3+ Treg and Th2 cells. While 2.5mi+ and GAD-specific T cells were tolerized in diabetes-resistant NODxB6.Foxp3EGFP F1 and nonobese resistant (NOR) mice, this did not occur in NOD mice. This indicated that NOD mice had a recessive genetic resistance to induce oral tolerance to both CTB-fused epitopes. In contrast to NODxB6.Foxp3EGFP F1 mice, oral treatment in NOD mice lead to strong 2.5mi+ T-cell activation and the sequestration of these cells to the effector-memory pool. Oral treatment of NOD mice with CTB-2.5mi failed to prevent diabetes. These findings underline the importance of investigating the effect of oral vaccine formulations on diabetogenic T cells as in selected cases they may have counterproductive consequences in human patients.