MicroRNAs are negative regulators of gene expression that play a key role in cell‐type specific differentiation and modulation of cell function and have been proposed to be involved in neovascularization. Previously, using an extensive cloning and sequencing approach, we identified miR‐126 to be specifically and highly expressed in human endothelial cells (EC). Here, we demonstrate EC‐specific expression of miR‐126 in capillaries and the larger vessels in vivo. We therefore explored the potential role of miR‐126 in arteriogenesis and angiogenesis. Using miR‐reporter constructs, we show that miR‐126 is functionally active in EC in vitro and that it could be specifically repressed using antagomirs specifically targeting miR‐126. To study the consequences of miR‐126 silencing on vascular regeneration, mice were injected with a single dose of antagomir‐126 or a control ‘scramblemir’ and exposed to ischemia of the left hindlimb by ligation of the femoral artery. Although miR‐126 was effectively silenced in mice treated with a single, high dose (HD) of antagomir‐126, laser Doppler perfusion imaging did not show effects on blood flow recovery. In contrast, quantification of the capillary density in the gastrocnemius muscle revealed that mice treated with a HD of antagomir‐126 had a markedly reduced angiogenic response. Aortic explant cultures of the mice confirmed the role of miR‐126 in angiogenesis. Our data demonstrate a facilitary function for miR‐126 in ischemia‐induced angiogenesis and show the efficacy and specificity of antagomir‐induced silencing of EC‐specific microRNAs in vivo.