AbstractGene therapy provides a new strategy for promoting endothelialization, and rapid endothelialization has attracted increasing attention for inhibiting thrombosis and restenosis in artificial vascular implants. However, the low transfection efficiency and high cytotoxicity of gene delivery systems prevent their in vivo application. In this study, an endothelial cell (EC)-specific gene carrier with relatively high transfection efficiency and low cytotoxicity was prepared successfully. Using bifunctional hydroxylsuccinimide–poly(ethylene glycol)–maleimide (NHS–PEG–MAL) as the linker, an EC-specific REDV peptide was conveniently grafted onto polyethyleneimine-b-poly(lactide-co-3(S)-methyl-morpholine-2,5-dione)-b-polyethyleneimine (PEI–PLMD–PEI). By varying the molar ratios of REDV to PEI, a series of REDV modified copolymers REDV–PEG-g-PEI–PLMD–PEI-g-PEG–REDV (REDV–PPP) were prepared. Then these copolymers were self-assembled into nanoparticles (NPs) as gene carriers. These NPs could easily condense the EGFP-ZNF580 plasmid (pZNF580) to form REDV peptide functionalized NP/pZNF580 complexes with low cytotoxicity. The fluorescence images, Western blot analysis, and quantitative real-time RT-PCR results verified that the effective transfection of REDV peptide functionalized NP/pZNF580 complexes in ECs was comparable with the positive control of PEI (25000 Da)/pZNF580 complexes. The high transfection efficiency was attributed to the enhanced cell uptake by the REDV peptide and relied on the quantity of the peptide. Furthermore, the rapid migration of the transfected ECs showed the active function of the expressed ZNF580 protein and further demonstrated that the REDV peptide functionalized NP/pZNF580 complexes could improve the transfection of pZNF580 in ECs. These results provided a useful platform to design EC-specific gene carriers and use gene therapy to enhance endothelialization.