A commercially available chitosan with a degree of deacetylation (DD) of 85% and a molecular weight (Mw) of 400 kDa was modified by acetylation with acetic anhydride to obtain a chitosan with a DD of 75%. Both polysaccharides were used to prepare DNA-chitosan nanoparticles by charge interactions with pDNA (coacervation process). Both resulting nanoparticles showed an almost total DNA loading efficiency (96%) and displayed similar physico-chemical properties with a size of similar to 200 nm and a zeta potential close to +20 mV. In order to study the effect of the DD on the properties of DNA-chitosan nanoparticles as gene delivery systems, the hydrodynamics-based procedure was used. The transgene expression was observed using either the green fluorescent protein (GFP) or the luciferase (Luc) as reporter genes. After the hydrodynamic injection, the DNA-chitosan nanoparticles were accumulated in the liver, where the transgene expression was mostly localized. Interestingly, the decrease of the DD affected the transgene expression, improving the initial burst effect and accelerating the DNA release. Both combined effects led to an increase in the transgene expression levels. In addition, the emitted bioluminescence could be detected over 105 days for all the formulations injected. The calculation of the kinetic parameters (C(max), AUC, Ke, t(1/2) Ke and MET) gave some interesting information regarding the abilities to control the DNA release of the two DNA-chitosan formulations tested and allowed narrower comparisons.