Mycobacterium tuberculosis (Mtb) must cope with exogenous oxidative stress imposed by the host. Unlike other antioxidant enzymes, Mtb’s thioredoxin reductase TrxB2 has been predicted to be essential not only to fight host defenses but also for in vitro growth. However, the specific physiological role of TrxB2 and its importance for Mtb pathogenesis remain undefined. Here we show that genetic inactivation of thioredoxin reductase perturbed several growth-essential processes, including sulfur and DNA metabolism and rapidly killed and lysed Mtb. Death was due to cidal thiol-specific oxidizing stress and prevented by a disulfide reductant. In contrast, thioredoxin reductase deficiency did not significantly increase susceptibility to oxidative and nitrosative stress. In vivo targeting TrxB2 eradicated Mtb during both acute and chronic phases of mouse infection. Deliberately leaky knockdown mutants identified the specificity of TrxB2 inhibitors and showed that partial inactivation of TrxB2 increased Mtb’s susceptibility to rifampicin. We also screened a library of 11,000 compounds with leaky knockdown mutants and identified SKF867J as a potential novel TrxB2-specific inhibitor. These studies reveal TrxB2 as an essential thiol-reducing enzyme in Mtb in vitro and during infection, establish the value of targeting TrxB2, and provide tools to accelerate the development of TrxB2 inhibitors. Ultimate control of Mtb is not achievable without effective vaccines. The most widely used tuberculosis (TB) vaccine, the Bacillus Calmette–Guérin (BCG) vaccine, does not provide effective protection against pulmonary TB. Current failures in TB vaccine development can be attributed in part to the lack of important virulence factors required to mediate protection in BCG-based vaccine candidates and insufficient antigen presentation at the site of infection. To overcome these limitations, we generated a novel Mtb-based vaccine candidate for proof-of-concept experiments, in which bacterial lysis is achieved by inducible expression of cell wall hydrolyzing enzymes, mycobacteriophage lysins. We found that lysin induction caused lytic death in both replicating and non-replicating Mtb. Inducible lysis restricted Mtb growth inside macrophages and enhanced the production of pro-inflammatory cytokines, possibly due to the release of intracellular bacterial antigens. Moreover, lysin induction impaired Mtb viability during mouse infection. We are now performing re-challenge experiments to determine the efficacy of the vaccine candidate against subsequent Mtb infection. Efforts are also underway to identify the immunological pathways activated by lysed bacteria and the bacterial components activating these pathways. In addition, we analyzed the sequences of 26 escape mutants of inducible lysis strains and showed that the tet repressor sequence is most frequently mutated. We are now designing new strains that combine other independent killing mechanisms to decrease the suppressor frequency.