Exosomes (EXOs) are secreted, nano-sized membrane vesicles that contain potent immunostimulatory materials. We have recently demonstrated that insulinoma-released EXOs can stimulate the autoimmune responses in nonobese diabetic (NOD) mice, a spontaneous disease model for type 1 diabetes. To investigate whether primary islet cells can produce EXOs, we isolated cells from the islet of Langerhans of NOD mice and cultured them in vitro. Interestingly, cultured islets release fibroblast-like, fast-replicating cells that express mesenchymal stem cell (MSC) markers, including CD105 and stem-cell antigen-1. These islet MSC-like cells release highly immunostimulatory EXOs that could activate autoreactive B and T cells endogenously primed in NOD mice. Serum EXO levels and EXO-induced interferon-γ production were positively correlated with disease progression at the early prediabetic stage. Consistent with these observations, immunohistological analysis of pancreata showed that CD105(+) cells are restricted to the peri-islet area in normal islets but penetrate into the β-cell area as lymphocyte infiltration occurs. Immunization with EXOs promoted expansion of transferred diabetogenic T cells and accelerated the effector T cell-mediated destruction of islets. Thus, EXOs could be the autoantigen carrier with potent adjuvant activities and may function as the autoimmune trigger in NOD mice.