Streptococcus pneumoniae is a Gram-positive bacterium which is a member of the normal human nasopharyngeal flora but can also cause serious disease such as pneumonia, bacteremia, and meningitis. Throughout its life cycle, S. pneumoniae is exposed to significant oxidative stress derived from endogenously produced hydrogen peroxide (H2O2) and from the host through the oxidative burst. How S. pneumoniae, an aerotolerant anaerobic bacterium that lacks catalase, protects itself against hydrogen peroxide stress is still unclear. Bioinformatic analysis of its genome identified a hypothetical open reading frame belonging to the thiol-specific antioxidant (TlpA/TSA) family, located in an operon consisting of three open reading frames. For all four strains tested, deletion of the gene resulted in an approximately 10-fold reduction in survival when strains were exposed to external peroxide stress. However, no role for this gene in survival of internal superoxide stress was observed. Mutagenesis and complementation analysis demonstrated that all three genes are necessary and sufficient for protection against oxidative stress. Interestingly, in a competitive index mouse pneumonia model, deletion of the operon had no impact shortly after infection but was detrimental during the later stages of disease. Thus, we have identified a gene complex involved in the protection of S. pneumoniae against external oxidative stress, which plays an important role during invasive disease. © 2012, American Society for Microbiology.