To explore the role of neutrophil-derived matrix metalloproteinases (MMPs) during angiogenesis in the brain, we hypothesized that transient neutrophil depletion attenuates the angiogenic response to focal hyperstimulation with vascular endothelial growth factor (VEGF). Brain focal angiogenesis was achieved using an adeno-associated virus delivered VEGF (AAV-VEGF) gene transfer in the mature mouse. Four groups of mice underwent AAV vector injection in the brain parenchyma: (1) AAV-LacZ; (2) AAV-VEGF; (3) AAV-VEGF plus anti-polymorphonuclear (PMN) antibody; and (4) AAV-VEGF plus serum. Animals in groups 3 and 4 underwent 4 days of PMN antibody or serum treatment before transfection; treatment was sustained for an additional 14 days. Anti-PMN treatment decreased circulating neutrophils to 9% of baseline (P < 0.001). Microvessels in the AAV-VEGF-group increased 25% compared with the AAV-lacZ-transduced group (256 ± 15 versus 208 ± 16; P < 0.05). Anti-PMN treatment attenuated the increase to 10% compared with control serum treatment (234 ± 16 versus 255 ± 22; P < 0.05). Similarly, compared with control serum treatment, anti-PMN treatment also reduced MMP-9 by 50% (2 ± 0.9 versus 4 ± 1.4; P < 0.05) and MPO expression by 25% (2 ± 0.8 versus 3 ± 0.9; P < 0.05); MMP-9 activity correlated with MPO expression (R² = 0.8, P < 0.05). Our study demonstrated that transient depletion of neutrophils suppressed VEGF-induced angiogenesis, indicating that circulating neutrophils contribute to VEGF-induced focal angiogenesis. In addition, brain MMP-9 activity was attenuated after neutrophil depletion, suggesting that neutrophil is an important source of MMP-9.