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&#945;-melanocyte-stimulating hormone (&#945;-MSH) has been characterized as a novel angiogenesis inhibitor. The homeostasis of nitric oxide (NO) plays an important role in neovascularization. However, it remains unclear whether &#945;-MSH mitigates angiogenesis through modulation of NO and its signaling pathway. The present study elucidated the function and mechanism of NO signaling in &#945;-MSH-induced angiogenesis inhibition using cultured human umbilical vein endothelial cells (HUVECs), rat aorta rings, and transgenic zebrafish. By Griess reagent assay, it was found &#945;-MSH dose-dependently reduced the NO release in HUVECs. Immunoblotting and immunofluorescence analysis revealed &#945;-MSH potently suppressed endothelial and inducible nitric oxide synthase (eNOS/iNOS) expression, which was accompanied with inhibition of nuclear factor kappa B (NF-&#954;B) activities. Excessive supply of NO donor <span style="font-variant: small-caps;">l</span>-arginine reversed the &#945;-MSH-induced angiogenesis inhibition in vitro and in vivo. By using antibody neutralization and RNA interference, it was delineated that melanocortin-1 receptor (MC1-R) and melanocortin-2 receptor (MC2-R) participated in &#945;-MSH-induced inhibition of NO production and NF-&#954;B/eNOS/iNOS signaling. This was supported by pharmaceutical inhibition of protein kinase A (PKA), the downstream effector of MC-Rs signaling, using H89 abolished the &#945;-MSH-mediated suppression of NO release and eNOS/iNOS protein level. Therefore, &#945;-MSH exerts anti-angiogenic function by perturbing NO bioavailability and eNOS/iNOS expression in endothelial cells.