Background: Mycothiol, the major low-molecular weight thiol of Mycobacterium tuberculosis, is important for peroxide detoxification and virulence. Results: Mycothiol, together with mycoredoxin-1, a glutaredoxin-like protein, reduces the one-cysteine peroxiredoxin AhpE from the bacterium. Conclusion: Mycobacterium tuberculosis AhpE is a mycothiol/mycoredoxin-1-dependent peroxidase. Significance: Our results provide the first molecular link between a thiol-dependent peroxidase and the mycothiol/mycoredoxin-1 pathway in Mycobacteria.