Mouse infection with intracellular bacteria induces a potent inflammatory response that requires protective mechanisms to avoid infection-induced immune pathology. CD69 is expressed in all leukocytes during activation after infection with a wide range of microbial pathogens. This study explores the way in which CD69 affects cell activation after Listeria monocytogenes (Lm) infection and its effects on host protection. We show that infectivity and bacterial clearance capability are unaltered in CD69(-/-) peritoneal macrophages, bone marrow-derived macrophages and dendritic cells. We found no major altered cell populations in splenocytes of Lm-infected CD69(-/-) mice. However, an increase in the expression of Th1 cytokines was observed after infection, with increased production of type I and II interferon (IFN). In addition, CD69(-/-) splenocytes showed increased apoptosis, consistent with IFN enhancement of lymphocyte apoptosis in response to Lm infection. CD69(-/-) mice showed liver and spleen damage, and greatly increased susceptibility to Lm infection, compared with wild-type controls. Lm-specific T cells were decreased in CD69(-/-) mice even if T-cell cross-presentation and T-cell intrinsic priming response were not compromised. As listeriosis was increased as early as day 1 post-infection but CD69(-/-)RAG2(-/-) mice were more efficient at controlling Listeria, we propose that CD69 controls the cross-talk between innate components and lymphocytes. These results highlight a role for CD69 in preventing infection-induced immunopathology.