Vascular endothelial growth factors (VEGFs) and their receptors have emerged as central regulators of the angiogenic process. However, involvement of VEGF-B, one of these factors, in angiogenesis remains obscure. Mice received subcutaneous injection of Matrigel alone or Matrigel with human recombinant protein rhVEGF-B₁₆₇ or with rhVEGF-A₁₆₅. After 14 days, cell ingrowth in the Matrigel plug was increased by 2.0- and 2.5-fold in rhVEGF-B₁₆₇–treated and rhVEGF-A₁₆₅–treated mice, respectively (P<0.01), in association with a raise in phospho-Akt/Akt (1.8-fold, P<0.01) and endothelial NO synthase (eNOS) (1.80- and 1.60-fold, respectively; P<0.05) protein levels measured by Western blot. VEGF-B–induced cell ingrowth was impaired by treatment with NOS inhibitor (N^G-nitro-l-arginine methyl ester; L-NAME, 10 mg/kg per day). Treatment with neutralizing antibody directed against the VEGF-B receptor VEGF-R1 (anti-VEGFR1, 10 μg) completely abrogated VEGF-B–related effects. Proangiogenic effect of VEGF-B was confirmed in a mouse model of surgically induced hindlimb ischemia. Plasmids containing human form of VEGF-A (phVEGF-A₁₆₅) or VEGF-B (phVEGF-B₁₆₇ or phVEGF-B₁₈₆) were administered by in vivo electrotransfer. Angiographic score at day 28 showed significant improvement in ischemic/nonischemic leg ratio by 1.4- and 1.5-fold in mice treated with phVEGF-B₁₆₇ and phVEGF-B₁₈₆, respectively (P<0.05). Laser Doppler perfusion data also evidenced a 1.5-fold increase in phVEGF-B₁₆₇–treated and phVEGF-B₁₈₆–treated mice (P<0.05). Such an effect was associated with an upregulation of phospho-Akt/Akt and eNOS protein levels in the ischemic legs and was hampered by treatment with anti-VEGFR1. This study demonstrates for the first time that VEGF-B, in part through its receptor VEGF-R1, promotes angiogenesis in association with an activation of Akt and eNOS-related pathways.