Abstract Rag-1–knockout (KO) mice are highly resistant to Listeria monocytogenes infection. The role played by the many Rag-1–dependent lymphocyte lineages was studied using a genetic approach in which each Rag-1–dependent lymphocyte lineage was eliminated one at a time. Only B cell-deficient Igh-KO mice displayed reduced bacterial load and improved survival upon Listeria infection. Listeria infection of Rag-1–KO and Il-10–KO hosts that had been adoptively transferred with wild-type marginal zone B (MZB) cells, but not follicular B cells, resulted in heightened bacterial load and increased Il-10 production in the spleen, but not the liver. This MZB cell-dependent increase in bacterial load was eliminated by anti–Il-10 mAb. In addition, Listeria infection of MZB cell-deficient Rbpj-cKO mice showed decreased bacterial load and increased survival. Whereas multiple cell types have been shown to be capable of Il-10 production, our results indicate that the MZB cell is the most dominant and relevant Il-10 source in the context of Listeria susceptibility. In marked contrast to the generally protective nature of MZB cells in defending against pathogenic infection, our results demonstrate that MZB cells play a detrimental role in Listeria infection and possibly other infections as well.