Left ventricular (LV) remodeling in heart failure (HF) is associated with vascular rarefaction and impaired angiogenesis. The inhibition of vascular endothelial growth factor (VEGF)-mediated angiogenesis is a key feature in the pathophysiology of HF. Semaphorin (Sema) 3F is a known inhibitor of VEGF signaling, but its role in HF remains to be elucidated. Serum Sema3F levels were measured in HF patients ( = 70) by ELISA and were compared to those in patients with coronary artery disease (CAD,  = 26). Sema3F levels were significantly increased in HF patients. Sema3F RNA and protein expression were upregulated by hypoxia in cardiac endothelial cells (HCECs) as demonstrated by quantitative RT-PCR and Western blotting (WB). In Matrigel® sprouting assays, endothelial cell sprouting and branching were decreased in response to HF patient serum, suggesting that HF serum contains anti-angiogenic factors. Recombinant human Sema3F attenuated VEGF-mediated angiogenesis in Matrigel® sprouting, spheroid sprouting and aortic ring assays. Vice versa, siRNA-based Sema3F knockdown promoted angiogenesis. In zebrafish, morpholino-based Sema3F knockdown led to increased mortality and induced a vascular phenotype. Mechanistically, Sema3F inhibited VEGF-induced Akt and eNOS phosphorylation in endothelial cells, and Sema3F knockdown increased phosphorylation of Akt and eNOS. Sema3F is elevated in serum of HF patients and has anti-angiogenic properties in cardiac angiogenesis through inhibition of the VEGF/Akt/eNOS pathway. Thus, targeting Sema3F could present a therapeutic approach to advanced HF in the future.