Type 1 diabetes (T1D) is an autoimmune disease characterized by T and B cell responses to proteins expressed by insulin-producing pancreatic β cells, inflammatory lesions within islets (insulitis), and β cell loss. We previously showed that Ag-specific tolerance targeting single β cell protein epitopes is effective in preventing T1D induced by transfer of monospecific diabetogenic CD4 and CD8 transgenic T cells to NOD. mice. However, tolerance induction to individual diabetogenic proteins, for example, GAD65 (glutamic acid decarboxylase 65) or insulin, has failed to ameliorate T1D both in wild-type NOD mice and in the clinic. Initiation and progression of T1D is likely due to activation of T cells specific for multiple diabetogenic epitopes. To test this hypothesis, recombinant insulin, GAD65, and chromogranin A proteins were encapsulated within poly(d,l-lactic- -glycolic acid) (PLGA) nanoparticles (COUR CNPs) to assess regulatory T cell induction, inhibition of Ag-specific T cell responses, and blockade of T1D induction/progression in NOD mice. Whereas treatment of NOD mice with CNPs containing a single protein inhibited the corresponding Ag-specific T cell response, inhibition of overt T1D development only occurred when all three diabetogenic proteins were included within the CNPs (CNP-T1D). Blockade of T1D following CNP-T1D tolerization was characterized by regulatory T cell induction and a significant decrease in both peri-insulitis and immune cell infiltration into pancreatic islets. As we have recently published that CNP treatment is both safe and induced Ag-specific tolerance in a phase 1/2a celiac disease clinical trial, Ag-specific tolerance induced by nanoparticles encapsulating multiple diabetogenic proteins is a promising approach to T1D treatment.