Alterations in ROS metabolism and redox signaling are often observed in cancer cells and play a significant role in tumor development and drug resistance. However, the mechanisms by which redox alterations impact cellular sensitivity to anticancer drugs remain elusive. Here we have identified the mitochondrial isoform of thioredoxin reductase 3 (mtTXNRD3), through RT-PCR microarray screen, as a key molecule that confers drug resistance to sorafenib and other clinical anticancer agents. High expression of mtTXNRD3 is detected in drug-resistant leukemia and hepatocellular carcinoma cells associated with significant metabolic alterations manifested by low mitochondrial respiration and high glycolysis. Mechanistically, high mtTXNRD3 activity keeps the mitochondrial thioredoxin2 (Trx2) in a reduced stage that in turn stabilizes several key survival molecules including HK2, Bcl-XL, Bcl-2, and MCL-1, leading to increased cell survival and drug resistance. Pharmacological inhibition of thioredoxin reductase by auranofin effectively overcomes such drug resistance in vitro and in vivo, suggesting that targeting this redox mechanism may be a feasible strategy to treat drug-resistant cancer.Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.