Gene therapy is the introduction of genes into cells or tissues to up- or down-regulate genes that can remove the pathological symptoms or activate the immune response via the delivery of DNA or RNA. This treatment has great promise for preventing or curing a wide range of diseases. In the present study, a RGD peptide and PEI grafted water soluble chitosan (RPgWSC) copolymer was synthesized and its efficiency as gene carrier investigated. The physiological characters, in vitro targeted gene transfection, cytotoxicity, blood-compatibility, and cellular distributions were investigated. In particular, a study of the endocytic mechanism resulted in the microtubule-dependent macropinocytosis and clathrin-mediated endocytosis.