The development of nonviral gene delivery vectors offers the potential to provide effective treatment for glioblastoma in the form of gene therapy. Here, we report the use of retro-inverso C-end rule (CendR) peptide (D)(RPPREGR) as a targeting ligand to prepare a (D)(RPPREGR)PEG-PEI gene vector. (D)(RPPREGR) peptide specifically recognized the neuropilin-1 receptor that was overexpressed on U87 glioma cells, and showed enhanced tumor spheroid penetration ability. Compared with parental RGERPPR, (D)(RPPREGR) possessed improved biological stability and had a higher affinity for U87 glioma cells; it also showed enhanced penetration of the tumor spheroid. mPEG-PEI/pDNA and (D)(RPPREGR)PEG-PEI/pDNA complexes were prepared and MTT assay results revealed that the cytotoxicity of (D)(RPPREGR)-PEG-PEI complexes was significantly lower than that of PEI complexes, with cell survival rates above 80%. Qualitative and quantitative in vitro transfection results revealed that (D)(RPPREGR)-PEG-PEI complex transfection efficiencies were 1.9 times higher than those of mPEG-PEI. Fluorescent imaging and frozen sections of brain tissue demonstrated that the (D)(RPPREGR) modification improved the in vivo transfection efficiency of mPEG-PEI in nude mice bearing U87 gliomas. An antiglioblastoma assay revealed that (D)(RPPREGR)-PEG-PEI carrying the therapeutic gene pORF-hTRAIL significantly prolonged the survival time of intracranial U87 glioma-bearing mice from 25 to 30 days. Therefore, (D)(RPPREGR)-PEG-PEI appears to be suitable for use as a safe and efficient gene delivery vehicle with potential applications in glioblastoma gene therapy.