Background: Success of any gene therapy protocol relies mostly on using an efficient carrier to direct nucleic acid to the place of action. The system should also have transfection ability at release site. Different routes are available for delivering genetic materials to the target organs, amongst them; oral delivery is particularly attractive for certain reasons. However, serious obstacles, like acidic environment of stomach and presence of protease and nuclease enzymes in gastrointestinal (GI) tract, make oral route a highly challenging option.Objectives: The present study suggests preparation of gene nanoparticles (NPs) of chitosan within a layer of Eudragit L100 for oral delivery of nucleic acid. The nanoparticles have some features both in size and polymer properties that can be penetrating enough to transfect epithelial layer cells of intestine and protect the entrapped materials against stomach harsh condition.Materials And Methods: In this experimental study, conducted in Iran, particles were prepared by coacervation technique followed by encapsulation of nanoparticle within a coat of Eudragit L100 using solvent evaporation technique. Formulation behavior was monitored both in vitro and in vivo. Stability of particle construction and release profile of DNA were examined at pH of ± 0.8 environ pKa of Eudragit. Size and zeta potential of particles were measured. To demonstrate transfection efficiency of the constructed carrier, reverse transcription polymerase chain reaction (RT-PCR) was carried out using human insulin specific primers on total RNA extracted from upper part of small intestine of 48-hour post-transfected rats (sampled by simple random selection, n = 3).Results: The mean size and zeta potential of particles were 300 ± 4 nm and 14 ± 0.5 mV, respectively. Encapsulation of this system was 89.6 ± 1.2%. DNA release from batches was less than 12% at pH = 5.2 and more than 60% at pH = 6.8 with significant difference of P < 0.05. RT-PCR product confirmed the presence of insulin transcript of 437 bp in upper intestinal extracts of the transfected rats. No band of DNA was seen after RT-PCR of placebo form of nanoparticles received group.Conclusions: Eudragit coated nanoparticle of chitosan is an efficient choice for oral delivery of DNA to upper part of GI tract.