Chitosan (CS) has been investigated as a non-viral carrier for gene delivery, but resulting in a relatively low transfection. To address this concern, we developed a ternary system comprised the core of CS/DNA complex and the outer coating of an anionic polymer, poly(γ-glutamic acid) (γ-PGA). In molecular dynamic (MD) simulations, we found that γ-PGA was entangle tightly with the excess CS emanating from the surface of test complexes, thus making them more compact. With γ-PGA coating, the extent of test complexes internalized and their transfection efficiency were evidently enhanced. Trypsin treatment induced a concentration-dependent decrease in internalization of the γ-PGA-coated complexes, suggesting a specific protein-mediated endocytosis. The endocytosis inhibition study indicates that the γ-glutamyl transpeptidase (GGT) present on cell membranes was responsible for the uptake of test complexes. The amine group in the N-terminal γ-glutamyl unit on γ-PGA played an essential role in the interaction with GGT. When entangled with CS, the free N-terminal γ-glutamyl unit of γ-PGA on test complexes was exposed and might thus be accommodated within the γ-glutamyl binding pocket of the membrane GGT. Above results suggest that the γ-PGA coating on CS/DNA complexes can significantly enhance their cellular uptake via a specific GGT-mediated pathway. Knowledge of the uptake mechanism is crucial for the development of an efficient vector for gene transfection. © 2012.