Significance Tissue antigen-specific Tregs are critical for the prevention of organ-specific autoimmune diseases, yet very little is known about their specificity in a natural repertoire. Here, we show an accumulation of antigen-experienced Tregs in the inflamed islets using a mouse model of autoimmune diabetes. MHC tetramer staining and single-cell T cell receptor (TCR) analyses of islet Tregs reveal their specificity for insulin and other islet-derived antigens. Tregs from inflamed islets prevent diabetes when transferred to diabetes-susceptible recipients. The identification of functional islet antigen-specific Tregs with distinct TCR usage paves the way for future investigations of their development and homeostasis to better understand pathogenesis and treatment of autoimmune diabetes.