Type 1 diabetes is an autoimmune disease in which autoreactive T cells attack and destroy the insulin-producing pancreatic β cells. CD8⁺ T cells are essential for this β cell destruction, yet their specific antigenic targets are largely unknown. Here, we reveal that the autoantigen targeted by a prevalent population of pathogenic CD8⁺ T cells in nonobese diabetic mice is islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Through tetramer technology, IGRP-reactive T cells are readily detected in islets and peripheral blood directly ex vivo. The human IGRP gene maps to a diabetes susceptibility locus, suggesting that IGRP also may be an antigen for pathogenic T cells in human type 1 diabetes and, thus, a new, potential target for diagnostic and therapeutic approaches.