Thyroid diseases are more prevalent in women, and this difference seems to be associated with the oxidative stress found in the thyroid of females. Thyroid NADPH Oxidase 4 (NOX4) was shown to respond to estrogen, which can also modulate TGF-β1, a potent stimulator of NOX4. This study aimed to investigate the effects of TGF-β1 on redox homeostasis parameters in the rat thyroid cell PCCL3 and the interrelationship between estrogen and TGF-β1. TGF-β1 treatment increased both intra- and extracellular ROS generation along with NOX4 expression and reduced GPX and catalase activities, extracellular H2O2 scavenging capacity, and reduced thiol content. TGF-β1 mRNA and protein expression are higher in female thyroid glands of rats in comparison to males. Moreover, 17β-estradiol treatment enhanced TGF-β1 mRNA in PCCL3 cells, decreased extracellular bioavailability but did not activate Smad pathway. Our data suggest that higher levels of TGF-β1 in females are potentially related to higher ROS availability which may be associated with the sex disparity in thyroid disorders.