Major research issues in protein delivery include the stabilization of proteins in delivery devices and the design of appropriate protein carriers in order to overcome mucosal barriers. We have attempted to combine both issues through the conception of new biodegradable polymer nanoparticles: (i) poly(ethylene glycol) (PEG)-coated poly(lactic acid) (PLA) nanoparticles, chitosan (CS)-coated poly(lactic acid-glycolic acid (PLGA) nanoparticles and chitosan (CS) nanoparticles. These nanoparticles have been tested for their ability to load proteins, to deliver them in an active form, and to transport them across the nasal and intestinal mucosae. Additionally, the stability of some of these nanoparticles in simulated physiological fluids has been studied. Results showed that the PEG coating improves the stability of PLA nanoparticles in the gastrointestinal fluids and helps the transport of the encapsulated protein, tetanus toxoid, across the intestinal and nasal mucosae. Furthermore, intranasal administration of these nanoparticles provided high and long-lasting immune responses. On the other hand, the coating of PLGA nanoparticles with the mucoadhesive polymer CS improved the stability of the particles in the presence of lysozyme and enhanced the nasal transport of the encapsulated tetanus toxoid. Finally, nanoparticles made solely of CS were also stable upon incubation with lysozyme. Moreover, these particles were very efficient in improving the nasal absorption of insulin as well as the local and systemic immune responses to tetanus toxoid, following intranasal administration. In summary, these results show that a rational modification in the composition and structure of the nanoparticles, using safe materials, increases the prospects of their usefulness for mucosal protein delivery and transport.