The development of safe, novel strong adjuvants is necessary to maximize the efficacy of and the immune response induced by new and/or available vaccines administered through the mucosal route to chickens. Chitosan is a non-toxic, biocompatible, biodegradable and natural polysaccharide derived from the exoskeleton of crustaceans and insects. It has been demonstrated to be an effective absorption enhancer to improve mucosal delivery of peptide and protein drugs in human and mice. In poultry, mucosal administration of live vaccine has been already explored with success. However, the effects of the use of the chitosan as adjuvant for mucosal vaccination in birds have not been investigated yet. To this aim, we explored its potential as adjuvant given by oculo-nasal route to one-day-old chickens with live Newcastle disease (ND) vaccine. The immune response has been evaluated during three independent vaccination experiments on specific pathogen free (SPF) chickens. It was shown that chitosan enhanced the antigen-specific cell-mediated immune response in the spleen. New protocols were developed to measure the chicken IFNgamma production after ex vivo antigen-stimulation of peripheral blood and duodenal lamina propria lymphocytes. It was then observed than the peripheral cellular immune response was earlier and stronger, while the local cellular immune response in digestive tract as shorter when chitosan was used as adjuvant. On the other hand, the chitosan had no effect on the systemic, lachrymal and digestive antibody-mediated immunity. This study indicates thus that the chitosan is a cell-promising adjuvant for the mucosal delivery of live vaccine in poultry, by enhancing the Th1 pathway of immunity. However, further investigations are required to explore its mechanism of action and to evaluate the inferred protection.