Campylobacter jejuni, a commensal Gram-negative motile bacterium commonly found in chickens is a frequent cause of human gastrointestinal infections. The polar flagellum of C. jejuni is an important virulence and colonization factor, providing motility to the cell as well as a type III secretion function. The flagellar biosynthesis genes fliA (sigma28) and rpoN (sigma54) of C. jejuni regulate a large number of genes involved in motility, protein secretion and invasion, which have been shown to be important factors for the virulence of this organism. To understand the role of the flagellar sigma factors, sigma28 and sigma54, in regulating colonization of the chicken intestinal tract, we assessed fliA and rpoN mutants of C. jejuni NCTC11168 for their ability to secrete Cia proteins and to adhere to and invade Hela cells. The mutants were also tested for their in vivo colonization potential in a chicken model with two different challenge doses. The fliA mutant showed reduced motility (25% that of the wild type) but secreted Cia proteins, yet it did not colonize the chicken cecum. The rpoN mutant cells lacked the spiral shape of C. jejuni and motility was reduced to 10% of the wild-type. The rpoN mutant did not secrete any Cia proteins but RT-PCR analysis showed the presence of ciaB mRNA, indicating that ciaB gene expression was independent of sigma54. Not surprisingly, the colonization defects of both fliA and rpoN mutants were more severe than the flgK mutant. We also demonstrated that FlgK, the hook filament junction protein of C. jejuni, is required for assembly of the flagellar secretory apparatus and an flgK mutant of C. jejuni expressing only the hook showed diminished motility and was completely attenuated for cecal colonization in chickens.