Cationic antimicrobial peptides (CAMPs) are an essential part of the innate immune system. Some Gram-negative enteric pathogens, such asSalmonella enterica, show intrinsic resistance to CAMPs. However, the molecular basis of intrinsic resistance is poorly understood, largely due to a lack of information about the genes involved. In this study, using a microarray-based genomic technique, we screened the Keio collection of 3,985Escherichia colimutants for altered susceptibility to human neutrophil peptide 1 (HNP-1) and identifiedenvCandzapBas novel genetic determinants of intrinsic CAMP resistance. In CAMP killing assays, anE. coliΔenvCEcor ΔzapBEcmutant displayed a distinct profile of increased susceptibility to both LL-37 and HNP-1. Both mutants, however, displayed wild-type resistance to polymyxin B and human β-defensin 3 (HBD3), suggesting that the intrinsic resistance mediated by EnvC or ZapB is specific to certain CAMPs. A correspondingSalmonellaΔenvCSemutant showed similarly increased CAMP susceptibility. TheenvCmutants of bothE. coliandS. entericadisplayed increased surface negativity and hydrophobicity, which partly explained the increased CAMP susceptibility. However, the ΔenvCEcmutant, but not the ΔenvCSemutant, was defective in outer membrane permeability, excluding this defect as a common factor contributing to the increased CAMP susceptibility. Animal experiments showed that theSalmonellaΔenvCSemutant had attenuated virulence. Taken together, our results indicate that the role ofenvCin intrinsic CAMP resistance is likely conserved among Gram-negative enteric bacteria, demonstrate the importance of intrinsic CAMP resistance for full virulence ofS. enterica, and provide insight into distinct mechanisms of action of CAMPs.