Distal skin ischemic necrosis is a common complication in skin flap surgery. The pathogenesis of skin flap ischemic necrosis is unclear and there is no clinical treatment available. Here, we used the 4x10cm rat dorsal skin flap model to test our hypothesis that subcutaneous injection of VEGF₁₆₅ in skin flaps at the time of surgery is effective in augmentation of skin flap viability which is associated with an increase in nitric oxide (NO) production and the mechanism involves: (1) an increase in skin flap blood flow in the early stage after surgery; and (2) enhanced angiogenesis subsequently to sustain increased skin flap blood flow and viability. We observed that subcutaneous injection of VEGF₁₆₅ in skin flaps at the time of surgery increased skin flap viability in a dose-dependant manner. Subcutaneous injection of VEGF₁₆₅ at the dose of 2µg/flap increased skin flap viability by 28% (p<0.05; n=8). Over 80% of this effect was blocked by intramuscular injection of the NO synthase (NOS) inhibitor N-nitro-L-arginine (13mg/kg) 45 min before surgery (p < 0.05; n=8). The VEGF₁₆₅ treatment also increased skin flap blood flow (2.68±0.63 ml/min/100g) compared with the control (1.26±0.10 ml/min/100g; p<0.05; n=6), assessed 6h postoperatively. There was no change in skin flap capillary density at this time-point. VEGF₁₆₅-induced increase in capillary density (32.2±1.1 capillaries/mm²; p<0.05, n=7) compared with the control (24.6±1.4 capillaries/mm²) was seen 7 days postoperatively. There was also evidence to indicate that VEGF₁₆₅-induced NO production in skin flaps was stimulated by activation of NOS activity followed by upregulation of NOS protein expression. These observations support our hypothesis and for the first time provide an important insight into the mechanism of acute local VEGF₁₆₅ protein therapy in mitigation of skin flap ischemic necrosis.