Campylobacter jejuni is the leading bacterial cause of human gastroenteritis in the United States. Increasing resistance of Campylobacter to clinical antibiotics raises an urgent need for novel strategies to prevent and control infections in humans and animal reservoirs, which necessitates a better understanding of Campylobacter pathogenesis. We hypothesize that multidrug efflux pump CmeABC and ferric enterobactin (FeEnt) iron acquisition systems, which play a critical role in Campylobacter pathogenesis, are novel targets for developing effective measures against Campylobacter. To test this, the molecular, antigenic, functional, and protective characteristics of two outer membrane proteins, CmeC (an essential component of CmeABC drug efflux pump) and CfrA (a FeEnt receptor), were examined. Both CmeC and CfrA are highly conserved and widely produced in C. jejuni strains. Anti-CmeC and Anti-CfrA antibodies inhibited the function of CmeABC efflux pump and CfrA, resulting enhanced susceptibility to bile salts and reduced utilization of FeEnt of C. jejuni, respectively. Immunoblotting analysis also indicated that CfrA is expressed and immunogenic in vivo. Amino acid substitution mutagenesis demonstrated that a highly conserved basic amino acid R327 in CfrA plays a critical role in FeEnt acquisition. The purified recombinant CmeC and a Salmonella live vaccine expressing the protective epitope of CfrA were evaluated as subunit vaccines against Campylobacter infection in the chicken model. CmeC vaccination elicited immune response but failed to reduce C. jejuni colonization in the intestine. However, Salmonella-vectored vaccine conferred significant protection against C. jejuni challenge. To further elucidate the role of iron acquisition in the pathogenesis of Campylobacter, whole genome sequence of a unique C. jejuni strain was determined using a 454 GS FLX sequencer with Titanium series reagents. Comparative genomics analysis led to the identification of a novel Campylobacter Enterobactin Esterase (Cee) that is essential in the CfrB-dependent FeEnt utilization pathway. Extensive genetic manipulation revealed molecular pathways and mechanistic features of the two orchestrated FeEnt acquisition systems in Campylobacter. This project provides critical information about the feasibility of targeting CmeC and CfrA for immune protection against Campylobacter colonization in the intestine, and increases our understanding of the critical role of FeEnt acquisition in the pathophysiology of Campylobacter.