In Neisseria gonorrhoeae, the MerR family transcription factor NmlR activates 3 operons in response to disulfide stress. In the present study, we show that trxB, a monocistronic operon under the control of NmlR, encodes a functional thioredoxin reductase. It is shown that neisserial TrxB has biochemical properties similar to those of its homologue from Escherichia coli. Analysis of a trxB mutant of N. gonorrhoeae showed that it was more sensitive to disulfide stress and to stress induced by organic hydroperoxides, superoxide, and nitric oxide than wild-type gonococcus. TrxB was found to be essential for the microaerobic induction of aniA and norB, the genes encoding nitrite reductase and nitric oxide reductase, respectively. The importance of TrxB during natural infection was demonstrated by the fact that the survival of gonococci within human cervical epithelial cells, as well as biofilm formation on these cells, was greatly reduced for a trxB mutant compared with a wild-type strain.