We previously developed chitosan (CS)-modified poly (D,L-lactide-co-glycolide) (PLGA) nanospheres (NS) by an emulsion solvent diffusion method as a gene delivery system. In this study, PLGA NS were modified using polysorbate 80 (P80) to improve their cellular uptake. We investigated the cellular uptake, intracellular distribution, and transfection efficiency of P80-modified PLGA NS (P80-PLGA NS) for a plasmid DNA delivery system in A549 cells. The cellular uptake and transfection efficiency of P80-PLGA NS were greater than CS-modified PLGA NS (CS-PLGA NS). The uptake of unmodified NS and CS-PLGA NS was mediated, predominantly, by clathrin-mediated endocytosis. In contrast, a specific endocytic pathway could not be determined for the cellular uptake of P80-PLGA NS. The intracellular distributions of PLGA NS depended on their surface properties. P80-PLGA NS were not cytotoxic for A549 cells. Thus, P80-PLGA NS could be used as an effective gene delivery system; the surface properties of PLGA NS are key parameters for optimal intracellular uptake and distribution.