Chitosan-pEGFP nanoparticles were synthesized through the complex coacervation of the cationic polymer with pEGFP, in order to examine the potential of chitosan as a non-viral gene delivery vector to transfer exogenous gene into primary chondrocytes for the treatment of joint diseases. The nanoparticles were prepared at an N/P ratio of 3.8 and showed a spherical or irregular shape. The mean particle size and zeta potential of the nanoparticles freshly prepared with chitosan of different molecular weight were in the range of 100–300nm and varied from +1 to +23mV, respectively. Both the particle size and the zeta potential altered in DMEM of different pH. The transfection of primary chondrocytes was performed in different conditions by varying pH of transfection medium, molecular weight of chitosan and different plasmid dosage. Analysis of FACS demonstrated that the transfection efficiency could reach a much high level and the percentage of positive cells could exceed 50% in certain condition. These results suggest that chitosan-DNA nanoparticles have favorable characteristics for non-viral gene delivery to primary chondrocytes, and have the potential to deliver therapeutic genes directly into joint.