Pathogenic mycobacteria including Mycobacterium tuberculosis resists phagocyte generated reactive oxygen intermediates (ROI) and this constitutes an important virulence mechanism. We have previously reported, using Mycobacterium smegmatis as a model to identify the bacterial components that resist intracellular ROI, that an antioxidant methionine sulfoxide reductase A (MsrA) plays a critical role in this process. In this study, we report the role of methionine sulfoxide reductase B (MsrB) in resistance to ROI by constructing a msrB mutant (MSΔmsrB) and MsrA/B double mutant (MSΔmsrA/B) strains of M. smegmatis and testing their survival in unactivated and interferon gamma activated mouse macrophages. WhilemsrB mutant exhibited significantly lower intracellular survival than its wild type counterpart, the survival rate seemed to be much higher than msrA mutant (MSΔmsrA) strain. Further, the msrB mutant showed no sensitivity to oxidants in vitro. The msrA/B double mutant (MSΔmsrA/B), on the other hand, exhibited a phenotype similar to that of msrA mutant in terms of both intracellular survival and sensitivity to oxidants. We conclude, therefore, that MsrB of M. smegmatis plays only a limited role in resisting intracellular and in vitro ROI.