Technical Abstract: Campylobacter jejuni is the most commonly reported cause of bacterial foodborne illness in North America. C. jejuni decorates its surface polysaccharides with a variety of variable phosphorylated structures, including O-methyl phosphoramidate (MeOPN) modifications on the capsular polysaccharide. Although MeOPN moieties are present in ~70% of C. jejuni isolates as well as several other members of the epsilonproteobacteria, little is known about their biological role. The C. jejuni genes involved in MeOPN biosynthesis and transfer to capsule have previously been identified in strain 11168, and our group has recently confirmed via site-directed mutagenesis that homologous genes in the highly virulent strain 81-176 are required for the biosynthesis and transfer of MeOPN. To determine the biological significance of MeOPN expression in C. jejuni, a variety of in vitro and in vivo assays were performed comparing the wild-type 81-176 strain to knockout mutants deficient in biosynthesis (cj1416) or transfer (orf7/orf20) of MeOPN. Mutants lacking MeOPN were more sensitive to serum and exhibited 10-fold greater invasion of Caco-2 epithelial cells. To investigate the potential insecticidal effects of the MeOPN structure in vivo, we used the Galleria mellonella (wax moth larvae) model system in which live C. jejuni bacterial cells are injected into the hemolymph of the larvae. In this model, wild type C. jejuni 81-176 efficiently killed G. mellonella in a dose-dependent manner and the cj1416 mutant displayed reduced killing, while complementation of this mutant resulted in wild-type killing. Interestingly, the orf7/orf20 mutant killed G. mellonella as efficiently as wild-type, suggesting that accumulation of MeOPN within the bacterial cells might be killing the insects. Infection experiments with piglets demonstrated that the cj1416 mutant was outcompeted by the wild-type strain. In contrast, the orf7/orf20 mutant showed similar levels of colonization as wild-type.