Present limitations in the management of extraocular diseases include the inability to provide long-term extraocular drug delivery without compromising intraocular structures and/or systemic drug exposure. In the present study, the potential of chitosan (CS) nanoparticles as a new vehicle for the improvement of the delivery of drugs to the ocular mucosa was investigated. Cyclosporin A (CyA) was chosen as a model compound because of its potential usefulness for the treatment of these local diseases. An ionic gelation technique was conveniently modified in order to produce CyA-loaded CS nanoparticles. These nanoparticles had a mean size of 293 nm, a zeta potential of +37 mV and high CyA association efficiency and loading (73 and 9%, respectively). In vitro release studies, performed under sink conditions, revealed a fast release during the first hour followed by a more gradual drug release during a 24-h period. In vivo experiments showed that, following topical instillation of CyA-loaded CS nanoparticles to rabbits, it was possible to achieve therapeutic concentrations in external ocular tissues (i.e., cornea and conjunctiva) during at least 48 h while maintaining negligible or undetectable CyA levels in inner ocular structures (i.e., iris/ciliary body and aqueous humour), blood and plasma. These levels were significantly higher than those obtained following instillation of a CS solution containing CyA and an aqueous CyA suspension. From these results, we can conclude that CS nanoparticles may represent an interesting vehicle in order to enhance the therapeutic index of clinically challenging drugs with potential application at extraocular level.