Transforming growth factor-β (TGF-β) is a cytokine that is profoundly related to tumorigenesis. We found that TGF-β stimulation increases the intracellular levels of reactive oxygen species (ROS) and alters the intracellular redox potential. In mammary gland epithelial cells treated with TGF-β, a mitochondria-dependent increase in the ROS concentration occurs in a sustained manner, while the mitochondrial membrane potential and intracellular glutathione (GSH) level decrease. To gain insights into the significance and mechanism of this response to TGF-β, we determined that approximately 15% of TGF-β-inducible genes are affected by mitochondrial depletion and therefore, we conducted further detailed analyses. Interestingly, we found that TGF-β-mediated expression of the epithelial–mesenchymal transition (EMT) marker fibronectin is inhibited by exogenously expressed mitochondrial thioredoxin (TXN2) independent of Smad signaling. Of note, the induction of high mobility group AT-hook 2 (HMGA2), a central mediator of EMT and metastatic progression, is similarly inhibited by TXN2 expression, revealing a novel mechanism involving a thiol oxidation reaction in mitochondria that regulates TGF-β-mediated gene expression associated with EMT.