Various laboratories have reported that local subcutaneous or subdermal injection of VEGF₁₆₅ at the time of surgery effectively attenuated ischemic necrosis in rat skin flaps, but the mechanism was not studied and enhanced angiogenesis was implicated. In the present study, we used the clinically relevant isolated perfused 6 × 16-cm pig buttock skin flap model to 1) test our hypothesis that VEGF₁₆₅ is a potent vasodilator and acute VEGF₁₆₅ treatment increases skin perfusion; and 2) investigate the mechanism of VEGF₁₆₅-induced skin vasorelaxation. We observed that VEGF₁₆₅ (5 × 10^–16–5 × 10^–11 M) elicited a concentration-dependent decrease in perfusion pressure (i.e., vasorelaxation) in skin flaps preconstricted with a submaximal concentration of norepinephrine (NE), endothelin-1, or U-46619. The VEGF₁₆₅-induced skin vasorelaxation was confirmed using a dermofluorometry technique for assessment of skin perfusion. The vasorelaxation potency of VEGF₁₆₅ in NE-preconstricted skin flaps (pD₂ = 13.57 ± 0.31) was higher (P < 0.05) than that of acetylcholine (pD₂ = 7.08 ± 0.24). Human placental factor, a specific VEGF receptor-1 agonist, did not elicit any vasorelaxation effect. However, a specific antibody to VEGF receptor-2 (1 μg/ml) or a specific VEGF receptor-2 inhibitor (5 × 10^–6 M SU-1498) blocked the vasorelaxation effect of VEGF₁₆₅ in NE-preconstricted skin flaps. These observations indicate that the potent vasorelaxation effect of VEGF₁₆₅ in the skin vasculature is initiated by the activation of VEGF receptor-2. Furthermore, using pharmacological probes, we observed that the postreceptor signaling pathways of VEGF₁₆₅-induced skin vasorelaxation involved activation of phospholipase C and protein kinase C, an increase in inositol 1,4,5-trisphosphate activity, release of the intra-cellular Ca²⁺ store, and synthesis/release of endothelial nitric oxide, which predominantly triggered the effector mechanism of VEGF₁₆₅-induced vasorelaxation. This information provides, for the first time, an important insight into the mechanism of VEGF₁₆₅ protein or gene therapy in the prevention/treatment of ischemia in skin flap surgery and skin ischemic diseases.