Although numerous determinants of L. monocytogenes cytosolic replication have been identified (14–16), few determinants of L. monocytogenes cytosolic survival are known (8, 17, 18). Thus, we designed and executed a novel genetic screen to identify L. monocytogenes mutants which lyse in the cytosol of macrophages. We identified mutations in genes regulating central metabolism and genes of unknown function critical for L. monocytogenes survival. Some genes were selectively required for survival in macrophages but not in other cell types, signifying cell type-specific cytosolic defenses. Unexpectedly, through an as-yet-undefined mechanism, a subset of mutants that lyse in the cytosol still avoided inflammasome activation. Despite this, all mutants identified in the screen were attenuated in a murine model of listeriosis. Next we investigated the function of menaquinone (MK) in cytosolic survival. We found that MK’s canonical functions in cellular respiration and the electron transport chain (ETC) were not critical for L. monocytogenes cytosolic survival. Instead, synthesis of the MK biosynthetic intermediate 1,4-dihydroxy-2-naphthoate (DHNA), but not of fully functional, isoprenylated menaquinone, was required for L. monocytogenes cytosolic survival. Taking the data together, our genetic screen uncovered factors required for L. monocytogenes survival in the host cytosol and evasion of the innate immune system and ultimately revealed a novel, ETC-independent function for DHNA. Additionally, these results add to the growing body of literature demonstrating that central metabolism plays a key role during host-pathogen interactions. Not only do host cells monitor and modulate their metabolism to sense and respond to pathogens (19), but cytosolic pathogens must also modulate their metabolism to avoid detection and/or killing by hosts.