How cytosolic pathogens avoid killing in the highly restrictive host cytosol is unknown. Using a novel genetic screen, we identified factors required for cytosolic survival of L. monocytogenes inside the host. Our approach uncovered genes involved in central metabolism and genes of unknown function required for the cytosolic survival and, ultimately, virulence of L. monocytogenes (see Table S1 in the supplemental material). Our data suggest that L. monocytogenes may have distinct strategies to overcome different nutritional limitations, stresses, and/or active CADs in different cytosolic environments (Fig. 1A; see also Fig. S3A and B in the supplemental material). Finally, we demonstrated that synthesis of the MK biosynthesis intermediate DHNA, but not of full-length isoprenylated MK, is essential for L. monocytogenes cytosolic survival in macrophages (Fig. 6B and E). Our findings identify some critical metabolic pathways for survival and immune evasion in the host cytosol. Finally, these findings are consistent with (and add to) recent studies demonstrating the intricate connection between pathogen and host metabolism in the context of pathogenesis and innate immune regulation.