Rationale: Vascular endothelial growth factor (VEGF) affects angiogenesis, atherosclerosis, and cancer. Although the heritability of circulating VEGF levels is high, little is known about its genetic underpinnings. Objective: Our aim was to identify genetic variants associated with circulating VEGF levels, using an unbiased genome-wide approach, and to explore their functional significance with gene expression and pathway analysis. Methods and Results: We undertook a genome-wide association study of serum VEGF levels in 3527 participants of the Framingham Heart Study, with preplanned replication in 1727 participants from 2 independent samples, the STANISLAS Family Study and the Prospective Investigation of the Vasculature in Uppsala Seniors study. One hundred forty single nucleotide polymorphism (SNPs) reached genome-wide significance ( P <5×10 ⁻⁸ ). We found evidence of replication for the most significant associations in both replication datasets. In a conditional genome-wide association study, 4 SNPs mapping to 3 chromosomal regions were independently associated with circulating VEGF levels: rs6921438 and rs4416670 (6p21.1, P =6.11×10 ⁻⁵⁰⁶ and P =1.47×10 ⁻¹² ), rs6993770 (8q23.1, P =2.50×10 ⁻¹⁶ ), and rs10738760 (9p24.2, P =1.96×10 ⁻³⁴ ). A genetic score including these 4 SNPs explained 48% of the heritability of serum VEGF levels. Six of the SNPs that reached genome-wide significance in the genome-wide association study were significantly associated with VEGF messenger RNA levels in peripheral blood mononuclear cells. Ingenuity pathway analyses showed found plausible biological links between VEGF and 2 novel genes in these loci ( ZFPM2 and VLDLR ). Conclusions: Genetic variants explaining up to half the heritability of serum VEGF levels were identified. These new insights provide important clues to the pathways regulating circulating VEGF levels.