Type 1 diabetes mellitus is caused by the autoimmune destruction of ${eta}$ cells within the islets. In recent years, innate immunity has been proposed to play a key role in this process. High-mobility group box 1 (HMGB1), an inflammatory trigger in a number of autoimmune diseases, activates proinflammatory responses following its release from necrotic cells. Our aim was to determine the significance of HMGB1 in the natural history of diabetes in non-obese diabetic (NOD) mice. We observed that the rate of HMGB1 expression in the cytoplasm of islets was much greater in diabetic mice compared with non-diabetic mice. The majority of cells positively stained for toll-like receptor 4 (TLR4) were ${eta}$ cells; few ${alpha}$ cells were stained for TLR4. Thus, we examined the effects of anti-TLR4 antibodies on HMGB1 cell surface binding, which confirmed that HMGB1 interacts with TLR4 in isolated islets. Expression changes in HMGB1 and TLR4 were detected throughout the course of diabetes. Our findings indicate that TLR4 is the main receptor on ${eta}$ cells and that HMGB1 may signal via TLR4 to selectively damage ${eta}$ cells rather than ${alpha}$ cells during the development of type 1 diabetes mellitus.