Novel heteroaromatic-substituted 4-hydroxycyclohexa-2,5-dienones (quinols) demonstrate potent in vitro antiproliferative activity and in vivo antitumor activity in tumor xenografts. The mechanism of action of these promising novel anticancer agents, however, remains to be fully elucidated. The thioredoxin (Trx) system comprising Trx, thioredoxin reductase (TrxR), and NADPH participates in a broad range of cellular functions involved in cell survival and proliferation. Accumulating evidence has indicated that the selenocysteine-containing mammalian TrxR is a valid molecular target for development of novel cancer therapeutics. In this study, we demonstrate that structural analogs containing a quinol pharmacophore inhibited TrxR with potencies correlated with their antiproliferative and cytotoxic efficacies. Benzenesulfonyl-6F-indole-substituted quinol (compound 6) irreversibly inhibited TrxR most strongly with a half-maximal inhibitory concentration of 2.7 µM after 1 h of incubation with recombinant rat TrxR. The inhibition was shown to be concentration-, time-, and NADPH-dependent and mediated through a direct quinol attack on the penultimate C-terminal selenocysteine residue. Moreover, TrxR activity in lysates of HCT 116 cells treated with apoptosis-inducing doses of quinols was significantly reduced. From the results obtained, we propose that TrxR inhibition is a critical cellular event that contributes to the proapoptotic effects of quinols.—Chew, E. H., Lu, J., Bradshaw, T. D., Holmgren, A. Thioredoxin reductase inhibition by antitumor quinols: a quinol pharmacophore effect correlating to antiproliferative activity.