Gene transfer is considered to be a promising alternative for the treatment of several chronic diseases that affect the ocular surface. The goal of the present work was to investigate the efficacy and mechanism of action of a novel DNA nanocarrier made of hyaluronan (HA) and chitosan (CS), specifically designed for topical ophthalmic gene therapy. With this goal in mind, we first evaluated the transfection efficiency of the plasmid DNA-loaded nanoparticles in a human corneal epithelium cell model. Second, we investigated the bioadhesion and internalization of the nanoparticles in the rabbit ocular epithelia by confocal laser scanning microscopy. Third, we determined the in vivo efficacy of these nanocarriers in terms of their ability to transfect ocular tissues. The results showed that HA-CS nanoparticles and, in particular, those made of low molecular weight CS (10-12 kDa), led to high levels of expression of secreted alkaline phosphatase in the human corneal epithelium model. In addition, we observed that, following topical administration to rabbits, these nanoparticles entered the corneal and conjunctival epithelial cells and, then, become assimilated by the cells. More importantly, these nanoparticles provided an efficient delivery of the associated plasmid DNA inside the cells, reaching significant transfection levels. Therefore, we conclude that these nanoparticles may represent a new strategy toward the gene therapy of several ocular diseases.