Objective: To investigate the protective effects of adenovirus-mediated vascular endothelial growth factor (Ad-VEGF) 165 gene transfer against hypoxic-ischemic brain damage (HIBD) in neonatal rats. Methods: Ad-VEGF recombinant adenovirus was constructed by bacterial homologous recombination technology. Seven-day-old Sprague-Dawley rats were randomly assigned to 4 groups: sham-operated (n = 20), HIBD (n = 25), buffer-treated (n = 20), and Ad-VEGF-treated (n = 25). The HIBD model was prepared by permanent occlusion of left common carotid artery, followed by exposure to 8 % oxygen for 2 hrs. In the Ad-VEGF-treated and the Buffer-treated groups, 2 μL recombinant adenovirus suspension or buffer was injected into the left sensorimotor cortex of the rat brain 3 days after HIBD. Seven days after transplantation, VEGF165 mRNA expression was detected using RT-PCR. Neuronal apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nickel end labeling (TUNEL). CD34 and VEGF protein were detected using immunohistochemistry. Microvascular density in the cerebral cortex was measured based on CD34 positive cells. A radial arm maze test was performed from 30 postnatal days to evaluate long-term learning and memory functions. At 35 postnatal days, the rats were sacrificed for cerebral histological examinations by hematoxylin and eosin. Results: The expression of VEGF165 mRNA increased in the Ad-VEGF-treated group more than in the untreated HIBD and the buffer-treated groups (P <0. 05). The number of apoptotic neurons was less in the Ad-VEGF-treated group compared with that in the untreated HIBD and the buffer-treated groups (P <0. 05). Microvascular density and VEGF positive cells increased in the Ad-VEGF-treated group compared with that in the untreated HIBD and the buffer-treated groups (P <0. 05). In the radial arm maze test, the Ad-VEGF-treated group had more improved achievements than the HIBD and the buffer groups (P <0. 05). Neuronal degeneration and necrosis were lessened in the Ad-VEGF-treated group compared with the HIBD and the buffer groups. Conclusions: Ad-VEGF gene transfer can increase the expression of VEGF mRNA and VEGF protein, decrease neuronal apoptosis, and increase angiopoiesis in the brain. This attenuates brain damage and improves long-term learning and memory functions in neonatal rats after HIBD.