Objective To investigate the feasibility of intramuscular gene therapy for acute arterial ischemic diseases by use of plasmid pcDNA3-VEGF121 and to evaluate therapeutic efficiency of vascular endothelial growth factor(VEGF) by different routes of administration. Methods Fifty New Zealand White rabbits were randomly assigned to either gelation sponge carryingpcDNA3-VEGF121 (n=18), intramuscular injectionpcDNA3-VEGF121 (n=18), or pcDNA3 (as control group,n=14). After ligation of the external iliac artery and complete excision of the femoral artery, 500 μg of the plasmid pcDNA 3-VEGF121 were transfected into the muscles of the ischemic limb by gelation sponge carrying or direct intramuscular-injection. Immediately after gene transfection, blood flow of the internal iliac artery were measured. VEGF121gene expression was detected by RT-PCR after 2 days, 1 week, 2 weeks, 3 weeks and 4 weeks of transfection. After 30 days, blood flow of the internal iliac artery, angiographic score and histologicalvessels of ischemic hindlimbs were measured respectively. Results In the two VEGF-treated groups, VEGF121 mRNA expressed in the transfected ischemic muscles after 2 days and lasted 2 weeks. Immediately after gene transfection, blood flow of the internal iliac artery had no significant difference between three groups. After 30 days, blood flow of the internal iliac artery, angiographicscore and capillary density were significantly greater in both VEGF-treated groups than in control group. Complexity of vascular branching and vessel density of gelation sponge-VEGF treated limbs were significantly greater when comparedwith the intramuscular-injection limbs. Conclusion These findings suggest the feasibility of employing gene therapy of pcDNA3-VEGF121could augmentcollatal development and tissue perfusion in an animal model of hindlimb ischemia, andgelation sponge carrying VEGF gene may respect a potential therapy methods.