Effective treatment of T1D requires a therapeutic strategy that can specifically target pathogenic T cells to reverse autoimmunity and rescue β-cell function without generalised immunosuppression. We have developed a preclinical mouse model of antigen-specific peptide immunotherapy (PIT), in which naturally processed and presented peptide fragments from major autoantigens are administered intradermally to specifically down modulate β-cell autoimmune responses. In these studies, HLA-DR4 Tg mice pre-treated with saline for 4 weeks before proinsulin-CFA immunisation generate robust anti-proinsulin autoimmune responses (proliferation, IFN-γ production). In contrast, pretreatment with proinsulin peptide for 4 weeks results in a down modulation of the proliferative and IFN-γ responses to whole proinsulin. Moreover, total cell numbers in the draining lymph nodes of proinsulin peptide treated mice are significantly reduced (p<0.05), suggesting an active regulatory process. Peptide-stimulated IL-10 levels are increased (p<0.05) and are unopposed by IFN-γ in the mice receiving active treatment, suggesting that a dominance of IL-10 is associated with tolerance in this model. To mirror more closely the clinical setting for its future use, we have also used this model to study PIT in the context of existing β-cell autoimmunity. Once proinsulin autoimmunity was established, HLA-DR4 Tg mice received saline or peptide for 4 weeks. Treatment resulted in a significant down modulation of the proliferative, IFN-γ and serum IgG responses to whole proinsulin (p<0.05), while the proportion of T-regs was significantly increased (p<0.05). We also used the model to examine the threshold (frequency of administration, amount of peptide per injection) of the effect. Treatment is more effective when peptide is administered frequently at low dose (p<0.05). This novel preclinical model of β-cell autoimmunity thus has important utility in aiding translation of antigen-specific immunotherapy into the clinical setting.