Bisphenol A (BPA), an endocrine disruptor, is widely used to manufacture polycarbonate plastic and epoxy resins. Many studies have demonstrated that BPA can play a role in reproductive toxicity and affect the normal metabolic function. Recent research has shown that BPA can influence the function of pancreatic islets. In this study, our aim is to assess the DNA damage induced by BPA and to clarify the mechanism, by use of rat insulinoma INS-1 cells. INS-1 cells were exposed to different doses of BPA (0, 25, 50, 100 μM). We conducted the single-cell gel electrophoresis (SCGE) assay to measure DNA damage, and studied proteins such as p53 and p-Chk2 (T68) by Western blotting, in order to verify the (geno)toxicity of BPA. Moreover, we examined intracellular reactive oxygen species (ROS) and glutathione (GSH) to discuss the possible mechanism of DNA damage. The results show that BPA caused an increased in DNA strand-breaks along with greater DNA migration from the nucleus into the comet tail. The expression of DNA damage-associated proteins (p53 and p-Chk2 (T68)) was significantly increased. The exposure to various doses of BPA caused a significant increase in intracellular ROS and a significant reduction in the level of GSH. N-Acetyl cysteine, an inhibitor of intracellular ROS formation, can significantly reduce the generation of intracellular reactive oxygen.