The thioredoxin (Trx) system, which consists of Trx and thioredoxin reductase (TrxR), is a major cellular disulfide reduction system important in antioxidant defense. TrxR is a target of mechlorethamine (methylbis(2-chloroethyl)amine; HN2), a bifunctional alkylating agent that covalently binds to selenocysteine/cysteine residues in the redox centers of the enzyme, leading to inactivation and toxicity. Mammalian Trx contains two catalytic cysteines; herein, we determined if HN2 also targets Trx. HN2 caused a time- and concentration-dependent inhibition of purified Trx and Trx in A549 lung epithelial cells. Three Trx cross-linked protein complexes were identified in both cytosolic and nuclear fractions of HN2-treated cells. LC-MS/MS of these complexes identified both Trx and TrxR, indicating that HN2 cross-linked TrxR and Trx. This is supported by our findings of a significant decrease of Trx/TrxR complexes in cytosolic TrxR knockdown cells after HN2 treatment. Using purified recombinant enzymes, the formation of protein cross-links and enzyme inhibition were found to be redox status-dependent; reduced Trx was more sensitive to HN2 inactivation than the oxidized enzyme, and Trx/TrxR cross-links were only observed using reduced enzyme. These data suggest that HN2 directly targets catalytic cysteine residues in Trx resulting in enzyme inactivation and protein complex formation. LC-MS/MS confirmed that HN2 directly alkylated cysteine residues on Trx, including Cys32 and Cys35 in the redox center of the enzyme. Inhibition of the Trx system by HN2 can disrupt cellular thiol-disulfide balance, contributing to vesicant-induced lung toxicity.