Campylobacter jejuni is the most common zoonotic bacterium associated with human diarrhea, and chickens are considered to be one of the most important sources for human infection, with no effective prophylactic treatment available. We describe here a prophylactic strategy using chitosan-DNA intranasal immunization to induce specific immune responses. The chitosan used for intranasal administration is a natural mucus absorption enhancer, which results in transgenic DNA expression in chicken nasopharynx. Chickens immunized with chitosan-DNA nanoparticles, which carried a gene for the major structural protein FlaA, produced significantly increased levels of serum anti-Campylobacter jejuni IgG and intestinal mucosal antibody (IgA), compared to those treated with chitosan-DNA (pCAGGS). Chitosan-pCAGGS-flaA intranasal immunization induced reductions of bacterial expellation by 2-3 log(10) and 2 log(10) in large intestine and cecum of chickens, respectively, when administered with the isolated C. jejuni strain. This study demonstrated that intranasal delivery of chitosan-DNA vaccine successfully induced effective immune response and might be a promising vaccine candidate against C. jejuni infection.