We have recently reported a new generation of polysaccharide nanoparticles consisting of chitosan (CS) and cyclodextrin (CD) derivatives, which exhibit a number of advantages when compared to the classical CS nanoparticles. In the present work our goal was to explore the potential of these hybrid CS/CD nanoparticles as carriers for the nasal delivery of macromolecules. First, we evaluated the effect of the amount and type of CD (sulfobutylether-beta-CD or carboximethyl-beta-CD) on the physicochemical properties of the nanocarriers. Second, we investigated the interaction of CS/CD nanoparticles with the nasal epithelium by studying their ability to modulate the tight junctions between epithelial cells (Calu-3 cell model) as well as their capacity to overcome mucosal barriers (nasal epithelium of rats). Finally, we loaded two selected nanocarriers with insulin and studied their potential for enhancing the nasal transport of insulin in rabbits. The results showed that CS/CD nanoparticles caused a reversible reduction in the transepithelial resistance of the cell monolayer, thus increasing the membrane permeability. Moreover, the results obtained following the in vivo administration of fluorescent CS/CD nanoparticles to rats evidenced their capability to overcome the nasal mucosal barrier. Finally, the in vivo evaluation in conscious rabbits revealed that insulin-loaded nanoparticles (association efficiencies > 88%) were able to significantly decrease plasma glucose levels (more than 35% reduction). Overall, these results suggest that these new nanoparticles work as nasal carriers and, therefore, have a potential for enhancing the transport of complex molecules across the nasal barrier.