Brain capillary endothelial cells (BCECs) have been considered as one of the primary targets for cerebral gene therapy. However, the cells, well-known for their poor function of endocytosis, are difficult to be transfected by general non-viral vectors. The aim of this study was to enhance the efficiency of transfection and expression in BCECs of DNA/polymer nanoparticles with the modification of membrane-penetrating peptide, Antennapedia peptide (Antp) polyethylenimine (PEI) and polyamidoamine (PAMAM) were chosen to prepare Antp-modified DNA-loaded nanoparticles with a complex coacervation technique. After a 20-min transfection, the efficiency, in terms of transfection and expression, of DNA/PEI NP or DNA/PAMAM NP was enhanced significantly with the modification of Antp. After a 3-h transfection of DNA/Antp/PEI NP, there was no difference in cellular uptake but an enhancement in gene expression, compared to DNA/PEI NP alone. However, both the transfection and expression efficiency of DNA/PAMAM NP were enhanced using Antp. These observations suggest that Antp can increase the membrane-penetrating ability of DNA-loaded nanoparticles, which can be employed as novel non-viral gene vectors.