MicroRNAs (miRs) 199a3p and 199a5p have been mainly implicated in proliferation, cell survival and remodeling in context of cancerogenesis but more recent data proposed a role in cardiovascular functions. We seek to assess how miR199a3p and miR199a5p modulate endothelial function by identifying their molecular targets in endothelial cells. Mice were treated with miRs199a3p/5p inhibitors (antagomiRs, 75mg/kg/day) by injection in the caudal vein and were sacrificed after 30 days. Contractile profile and endothelial function were evaluated ex vivo in the aorta. Cultured endothelial cells (Sigma) were transfected with miRs199a3p/5p inhibitors (locked-nucleic acid (LNA)) or a scramble sequence. NO production, expression and/or activity of endothelial nitric oxide synthase (eNOS) and its major regulators were measured by electroparamagnetic resonance (EPR) and Western blot respectively. Implication of protein kinase B (Akt or PKB) pathway was analysed using LY294002 treatment (20µM) or Akt1 small interferent RNA (siRNA, 50nM). Angiogenesis was investigated in 2D-culture in matrigel support. Vessels from mice treated with antagomirs against miR199a3p or 5p showed a larger NO-dependent relaxation compared to controls (ANOVA: p<0.001 ctl 37,4±4.2 (n=4) vs antagomiR a3p: 72.6±2.8 (n=5) and antagomiR a5p: 69.0±8.8 (n=5) maximal relaxation). Circulating haemoglobin-NO measured by EPR in venous blood collected from these mice was significantly increased compared to controls (ANOVA, Tukey-Kramer post-test: p<0.05, ctl 100±17% (n=4) vs antagomiR a3p: 339±59% (n=5) and antagomiR a5p: 240±41.2% (n=5)) suggesting a role of both miRs in the control of the NOS/ NO pathway. Repression of miR 199a3p/5p improved eNOS activity through an increased eNOS phosphorylation at serine 1177 (N=6) and a decreased phosphorylation status of threonine 495 (N=5). The eNOS allosteric regulator, caveolin-1, was not modulated by LNA treatments (N=3). Both treatments promoted calcineurin expression (N=4) and Akt activation (N=5). Furthermore, addition of LY294002, a Akt inhibitor (N=3), or specific knockdown of Akt1 by siRNA (N=3), inhibited the increase of eNOS phosphorylation at serine 1177 induced by LNA treatment. Interestingly, repression of miR199a5p promoted superoxide dismutase (SOD) expression (N=3), suggesting that miR199a5p also improved NO bioavailability. In addition, inhibition of miR199a5p upregulated vascular endothelial growth factor (VEGF) production and promoted tubes formation in bovine endothelial cells (N=3). Our results demonstrate that miRs199a3p/5p modulate the NOS/NO pathway in the endothelium by promoting NO production and repressing NO degradation. These results also suggest a strong implication of the Akt pathway in this modulation. Interestingly, miR199a5p shows additional control of angiogenesis.