The goal of the present work was to develop a new synthetic nanosystem for gene delivery. For this purpose, we chose two polysaccharides, hyaluronic acid (HA) and chitosan (CS), as the main components of the nanocarrier. Nanoparticles with different hyaluronate:chitosan (HA:CS) mass ratios (0.5:1 and 1:1) and different polymer molecular weights (hyaluronate 170 (HA) or <10 kDa (HAO) and chitosan 125 (CS) or 10-12 (CSO) kDa) could be obtained using an ionic crosslinking method. These nanoparticles were loaded with pDNA and characterized for their size, zeta potential and pDNA association efficiency. Moreover, their toxicity and ability to transfect the model plasmid pEGFP-C1 were evaluated in the cell line HEK 293, as well as their intracellular fate. The results showed that HA:CS nanoparticles have a small size in the range of 110-230 nm, a positive zeta potential of +10 to +32 mV and a very high pDNA association efficiency of 87-99% (w/w). On the other hand, nanoparticles exhibited low cell toxicity and transfection levels up to 25% GFP expressing HEK 293 cells, lasting for the whole observation period of 10 days. We also provide basic information about the role of both polymers, HA and CS, and the effect of their molecular weight on the effectiveness of the resulting DNA nanocarrier, being the highest transfection levels observed with HAO:CSO 1:1 nanoparticles. In conclusion, HA:CS nanoparticles are promising carriers for gene delivery.