Salmonella enterica serovar Choleraesuis (SC) is an important enteric pathogen that causes serious systemic infections in swine and humans. To identify the genes required for resistance to antimicrobial peptides, we constructed a bank of SC transposon mutants and screened them for hypersensitivity to the cationic peptide polymyxin B. Here we report one isolated polymyxin B-susceptible mutant that also exhibited increased sensitivity toward human neutrophil peptide alpha-defensin 1 (HNP-1) and hydrophobic antibiotics including erythromycin and novobiocin. The mutant had a mutation in an ORF identified as outer membrane beta-barrel protein gene omb. The purified recombinant Omb protein was characterized as a ferrous iron-binding protein. The constructed omb isogenic mutant grew more slowly in iron-limiting conditions than the wild-type (WT) parent strain. In addition, compared with the WT strain, the omb mutant exhibited an increase in net negative charge upon the cell surface and was more easily killed by polymyxin B, HNP-1 and hydrophobic antibiotics. The omb gene was transcribed, regardless of the iron content within the growth medium, and the Climb protein appeared exclusively in the outer membrane fraction. Infection experiments demonstrated virulence attenuation when the mutant was administered orally or intraperitoneally to mice. This study indicates that Climb is a previously unrecognized ferrous iron-binding protein. In vivo, Omb may be involved in the acquisition of ferrous iron during the initial stages of SC infection and appears to be an important virulence factor for SC in mice.