DNase I is an endonuclease responsible to destruction of chromatin during apoptosis. However, its role in diabetes is still unclear. With blood samples from our previous study related to type 2 diabetes, we examined the DNase I activity in the serum of these patients and the role of DNase I in the injury of pancreas was further investigated in rats and INS-1 cells. Serum and pancreatic tissues from human and rats were used for the study. Insulin resistance and diabetes were induced by high fat diet and STZ injection, respectively. DNase I activity was determined by radial enzyme-diffusion method. Expressions of DNase I and caspase-3 in pancreas were determined in rat pancreatic tissues and INS-1 cells. Apoptosis of INS-1 cells was determined by both TUNEL assay and Flow Cytometry. There was a significant elevation of DNase I activity in serum of patients with type 2 diabetes and rats with STZ injection. Moreover, increase in DNase I expression was observed in the pancreas of diabetic person and rats. Furthermore, high glucose induced both DNase I and caspase-3 expression and at the same time increased apoptosis rate of INS-1 cells. In conclusion, elevated DNase I in diabetes may be related to pancreatic injury and could be one of the causes that induce diabetes.