Critical limb ischemia is regarded as a potentially lethal disease, and the treatment effects of existing therapies are limited. Here, in order to develop a potential approach to improve the therapy effects, we designed a peptide of TAT-PKKKRKV as the vector for VEGF165 plasmid to facilitate in vivo angiogenesis. In in vitro studies, TAT-PKKKRKV with low cytotoxicity exhibited efficient transfection ability either with or without serum. Additionally, application of TAT-PKKKRKV/VEGF165 complexes in hindlimb ischemia rats obviously promoted the expression of VEGF protein, which further enhanced effective angiogenesis. The results indicated that TAT-PKKKRKV is an efficient gene vector with low toxicity both in vitro and in vivo, which has great potential for clinical gene therapy.