We investigated the interactions between Thioredoxin (Trx) and BNP7787 (Tavocept®). BNP7787 is a water-soluble disulfide that appears well-tolerated and nontoxic, and in separate randomized multicenter Phase 2 and Phase 3 clinical trials in NSCLC patients, BNP7787 in combination with standard chemotherapy, resulted in substantial increases in the overall survival of patients with advanced adenocarcinoma sub-type NSCLC. Activity assays indicate that BNP7787 and BNP7787-derived mesna disulfide heteroconjugates are novel alternative substrates of Trx with the potential to inhibit turnover of endogenous Trx substrates. LC-MS studies indicate that BNP7787 forms a covalent mixed-disulfide with Trx on cysteine residues 62, 69 and 73. BNP7787-modified Trx is less active than apo-Trx. X-ray studies unequivocally confirm that BNP7787 forms stable mixed disulfides with Trx. BNP7787-modified Trx crystallized as a disulfide-linked tetramer with novel interactions between BNP7787-derived mesna and Trx, including stable BNP7787-derived mixed mesna-cysteine disulfides on human Trx resulting in unique, previously unidentified Trx structural conformations. Molecules A and B of the tetramer are identical to each other and exist in a conformation similar to other published Trx crystals. Molecules C and D, also identical to each other, undergo a significant conformational change relative to molecules A and B, and exhibit substantial disulfide reshuffling. Inhibition or modulation of Trx by BNP7787 through covalent or non-covalent means may have important ramifications in cancers with elevated Trx activity, including adenocarcinoma sub-type NSCLC, and could impact Trx-related signaling pathways. important for the inhibition of apoptosis and cell proliferation.