Translational readthrough, observed primarily in less complex organisms from viruses to Drosophila, expands the proteome by translating select transcripts beyond the canonical stop codon. Here, we show that vascular endothelial growth factor A (VEGFA) mRNA in mammalian endothelial cells undergoes programmed translational readthrough (PTR) generating VEGF-Ax, an isoform containing a unique 22-amino-acid C terminus extension. A cis-acting element in the VEGFA 3' UTR serves a dual function, not only encoding the appended peptide but also directing the PTR by decoding the UGA stop codon as serine. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 binds this element and promotes readthrough. Remarkably, VEGF-Ax exhibits antiangiogenic activity in contrast to the proangiogenic activity of VEGF-A. Pathophysiological significance of VEGF-Ax is indicated by robust expression in multiple human tissues but depletion in colon adenocarcinoma. Furthermore, genome-wide analysis revealed AGO1 and MTCH2 as authentic readthrough targets. Overall, our studies reveal a novel protein-regulated PTR event in a vertebrate system.