Mammalian thioredoxin reductase (TR) is a selenocysteine (Sec)-containing homodimeric pyridine nucleotide oxidoreductase which catalyzes the reduction of oxidized thioredoxin. We have previously demonstrated the full-length mitochondrial mammalian TR (mTR3) enzyme to be resistant to inactivation from exposure to 50 mM H2O2. Because a Sec residue oxidizes more rapidly than a cysteine (Cys) residue, it has been previously thought that Sec-containing enzymes are "sensitive to oxidation" compared to Cys-orthologues. Here we show for the first time a direct comparison of the abilities of Sec-containing mTR3 and the Cys-orthologue from D. melanogaster (DmTR) to resist inactivation by oxidation from a variety of oxidants including H2O2, hydroxyl radical, peroxynitrite, hypochlorous acid, hypobromous acid, and hypothiocyanous acid. The results show that the Sec-containing TR is far superior to the Cys-orthologue TR in resisting inactivation by oxidation. To further test our hypothesis that the use of Sec confers strong resistance to inactivation by oxidation, we constructed a chimeric enzyme in which we replaced the active site Cys nucleophile of DmTR with a Sec residue using semisynthesis. The chimeric Sec-containing enzyme has similar ability to resist inactivation by oxidation as the wild type Sec-containing TR from mouse mitochondria. The use of Sec in the chimeric enzyme "rescued" the enzyme from oxidant-induced inactivation for all of the oxidants tested in this study, in direct contrast to previous understanding. We discuss two possibilities for this rescue effect from inactivation under identical conditions of oxidative stress: (i) Sec resists overoxidation and inactivation, whereas a Cys residue can be permanently overoxidized to the sulfinic acid form, and (ii) Sec protects the body of the enzyme from harmful oxidation by allowing the enzyme to metabolize (turnover) various oxidants much better than a Cys-containing TR.