The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2 and proinsulin, in exosomes, which are taken up by and activate dendritic cells. Accordingly, anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T cell activation in the context of the human type 1 diabetes susceptibility haplotype HLA-DR4. Cytokine-induced ER-stress enhanced exosome secretion by β-cells, induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96 and ORP150 and increased exosomal stimulation of antigen presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in initiation of autoimmune responses in T1D. © 2016 by the American Diabetes Association.