Diabetes mellitus (DM) disrupts the pituitary-thyroid axis and leads to a higher prevalence of thyroid disease. However, the role of reactive oxygen species in DM thyroid disease pathogenesis is unknown. Dual oxidases (DUOX) is responsible for H(2)O(2) production, which is a cosubstrate for thyroperoxidase, but the accumulation of H(2)O(2) also causes cellular deleterious effects. Nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) is another member of the nicotinamide adenine dinucleotide phosphate oxidase family expressed in the thyroid. Therefore, we aimed to evaluate the thyroid DUOX activity and expression in DM rats in addition to NOX4 expression. In the thyroids of the DM rats, we found increased H(2)O(2) generation due to higher DUOX protein content and DUOX1, DUOX2, and NOX4 mRNA expressions. In rat thyroid PCCL3 cells, both TSH and insulin decreased DUOX activity and DUOX1 mRNA levels, an effect partially reversed by protein kinase A inhibition. Most antioxidant enzymes remained unchanged or decreased in the thyroid of DM rats, whereas only glutathione peroxidase 3 was increased. DUOX1 and NOX4 expression and H(2)O(2) production were significantly higher in cells cultivated with high glucose, which was reversed by protein kinase C inhibition. We conclude that thyroid reactive oxygen species is elevated in experimental rat DM, which is a consequence of low-serum TSH and insulin but is also related to hyperglycemia per se.