Gene therapy provides a promising treatment for glioblastoma multiforme, which mainly depends on two key aspects, crossing the blood brain barrier (BBB) effectively and transfecting target cells selectively. In this work, we reported a series of peptide-based vectors for transfecting glioma cells specifically consisting of several functional segments including a cell-penetrating peptide, targeting segment substance P (SP), an endosomal escape segment, a PEG linker and a stearyl moiety. The conformations and DNA-loading capacities of peptide vectors and the self-assembly behaviors of peptide/pGL3 complexes were characterized. The in vitro gene transfection was evaluated in U87, 293T-NK1R, and normal 293T cell lines. The transfection efficiency ratio of P-02 (SP-PEG4-K(C18)-(LLHH)3-R9) to Lipo2000 in the U87 cell line was about 36% higher than that in the 293T cell line. The neurokinin-1 receptor (NK1R) in U87 cells mediated the transfection process via interactions with the ligand SP in peptide vectors. The mechanism of NK1R mediated transfection was demonstrated by the use of gene-modified 293T cells expressing NK1R, as well as the gene transfection in the presence of free SP. Besides, P-02 could promote the pGL3 plasmids to cross the BBB model in vitro and achieved the EGFP gene transfection in the brain of zebrafish successfully. The designed peptide vectors, owing to their specific transfection capacity in glioma cells, provide a potential approach for glioblastoma multiforme gene therapy.