Background: Although reactive oxygen species (ROS) are integral for TGF-␤ signaling, the source of ROS is not clear. Results: Inhibition of TGF-␤-induced mitochondrial ROS generation attenuates profibrotic gene expression. Conclusion: ROS generated by complex III of the electron transport chain are required for TGF-␤-mediated transcription in normal human lung fibroblasts. Significance: Mitochondrial ROS might be a novel target to prevent TGF-␤-mediated induced fibrosis.