Worldwide research devotes significant effort to identify new, targetable molecular mechanisms in the field of angiogenesis, since therapeutic modulation of angiogenesis can critically alter the progression of a number of diseases. Stimulation of angiogenesis is desirable in situations characterized by tissue-damaging ischemia where blood supply is severely reduced, such as lower limb diabetic arteriopathy or following myocardial infarct. In contradistinction, stemming excessive or ectopic angiogenesis can be beneficial in situations such as solid tumor growth or in neovascular age-related macular degeneration. It has been previously shown that Hydrogen Sulfide (H2S), a new vasoactive gasotransmitter, can initiate angiogenic responses which depend on the activation of ATP-sensitive potassium channels (KATP). Intriguingly, C-type Natriuretic Peptide (CNP), which is also known to activate KATP, has been reported to promote endothelial cell growth; however, its angiogenic properties have not been explored at any depth. This pattern prompted us to investigate whether direct KATP activation induces angiogenic responses and whether endothelial responses to CNP or Vascular Endothelial Growth Factor (VEGF) indeed require KATP activation. We undertook a dual-pronged approach, based on both in vivo and in vitro experimental approaches. In vivo, chick embryo chorioallantoic membrane (CAM) angiogenesis was similarly enhanced by the direct KATP channel activator SG-209 and by the polypeptides CNP or VEGF. Two KATP inhibitors, Glibenclamide and 5-Hydroxydecanoate (5-HD), abrogated both basal and CNP-induced CAM angiogenesis. In vitro, direct activators of KATP such as Nicorandil and SG- 209 and receptor-acting agonists such as VEGF and CNP, increased proliferation and migration in the mouse brain endothelial cell line bEnd.3. In addition, VEGF and CNP induced comparable capillary tube-like formation by Human Umbilical Vein Endothelial cells (HUVECs) in low growth factor Matrigel. All these in vitro pro-angiogenic endothelial responses were effectively abrogated by Glibenclamide or by 5-HD. Transfection of HUVECs with a siRNA specifically targeting the inwardly rectifying potassium channel (Kir) 6.1 subunit decreased the expression of this subunit at both the mRNA and the protein level. The resulting knock-down of the Kir6.1 KATP subunit impaired HUVEC migration through transwells in vitro and substantially decreased tubular network formation in Matrigel in response to either the direct KATP activator SG-209 or the receptor-operating KATP activator CNP. Furthermore, the bEnd.3 endothelial cell proliferation and migration responses to SG-209 required mobilization of the “classic” endothelial pro-angiogenesis kinases Erk1/2, p38 and Akt, since the responses to SG-209 were all abolished by each of the respective kinase inhibitors. This work allows us to firmly conclude that: a) direct pharmacological modulation of KATP channels affects angiogenic responses in vitro and in vivo, b) CNP is a bona fide angiogenic factor, as potent and efficient to mobilize endothelial cells as VEGF, c) the angiogenic effects of CNP and VEGF depend on the activation of endothelial KATP channels and specifically require the expression of the Kir6.1 pore-forming KATP subunit, and finally d) KATP activation may be a common molecular mechanism that underpins angiogenesis to a wide variety of endogenous vasoactive stimuli that includes H2S, VEGF and CNP. The therapeutic implications of this work are significant: Sulfonylurea-type KATP channel inhibitors, with questionable selectivity for pancreatic β-cells, are widely used drugs to treat type II diabetes, a disease characterized by arterial dysfunction and higher incidence of myocardial and lower limb ischemia. The outcome of cardiac ischemia in diabetic patients is worse if they have been treated with sulfonylureas, indicating some, until now unresolved, deleterious cardiovascular activity of this class of compounds. The present demonstration that endothelial KATP channel activation is a common pro-angiogenic mechanism, may in part explain this unfavorable outcome of sulfonylurea treatment in diabetics. Furthermore, it raises the need to design new molecules which, while inhibiting the pancreatic KATP channels, should spare the endothelial KATP channels and the ensuing angiogenesis, thus exhibiting increased therapeutic benefit.