Different factors are involved in thyroid function and proliferation such as thyrotropin (TSH), insulin, growth factors, iodide, etc. TSH and IGF1/insulin increase proliferation rate and stimulate genes involved in thyroid differentiation. In the present study, we analyse the physiological regulation of NOX4 expression by TSH, insulin and iodine, and the role of NOX4 on thyroid genes expression. Differentiated rat thyroid cells (FRTL-5) were incubated in the presence or absence of TSH/insulin and TTF2, PAX8, TPO, NIS, NOX4, TGFβ1, FOXO1/3 mRNA levels were examined by Real Time PCR. We showed that TSH and insulin repress NOX4 expression and appears to be inversely correlated with some thyroid genes. SiRNA targeted knockdown of NOX4 increased mRNA levels of TGFβ1, TPO, PAX8, TTF2, FOXO1 and FOXO3. A PI3K inhibitor (LY294002), increases the expression of NIS, TTF2 and FOXO1/3, however PI3K/AKT pathway does not regulate NOX4 expression. We observed that iodine increased NOX4 expression and knockdown of NOX4 reduced ROS and reversed the inhibitory effect of iodine on NIS, TPO, PAX8 and TTF2 expression. Our findings provide strong evidence that NOX4 could be a novel signaling modulator of TSH/insulin pathway and would have a critical role in the autoregulatory mechanism induced by iodine.