This work investigated polyethyleneimine (PEI)-introduced chitosan (CS) (CS/PEI) nanoparticles as the non-viral carriers of plasmid DNA for rat mesenchymal stem cells (MSC). The CS/PEI nanoparticles were prepared by the emulsifier-free emulsion polymerization of methyl methacrylate monomer induced by small amount of t-butyl hydroperxide in the presence of different concentrations of PEI mixed with CS. The resulted nanoparticles were characterized by the surface properties, and buffering capacity. In vitro gene transfection was also evaluated. The introduction of PEI affected the surface charge, dispersing stability, and buffering capacity of the nanoparticles. The CS/PEI nanoparticles formed a complex upon mixing with a plasmid DNA of luciferase. The complex enhanced the level of gene transfection and prolonged the time period of expression for MSC, compared with those of plasmid DNA-original CS and PEI nanoparticles. Cytotoxicity of CS/PEI complexes with plasmid DNA was significantly low, however, depending on the amount of PEI introduced. It is concluded that the CS/PEI nanoparticle was a promising carrier for gene delivery of MSC.