Recent studies have shown that a carbohydrate-binding protein, galectin-3, is a novel pro-angiogenic molecule. The mechanism by which galectin-3 promotes angiogenesis remains unknown. We demonstrate here that galectin-3 is a mediator of vascular endothelial growth factor (VEGF)- and basic fibroblast growth factor (bFGF)-mediated angiogenic response. Angiogenesis assays revealed that galectin-3 inhibitors, beta-lactose and dominant-negative galectin-3, reduce VEGF- and bFGF-mediated angiogenesis in vitro and that VEGF- and bFGF-mediated angiogenic response is reduced in galectin-3 knockdown cells and Gal3(-/-) animals. Integrin alphavbeta3 was identified as the major galectin-3-binding protein and anti-alphav, -beta3, and -alphavbeta3 integrin function-blocking antibodies significantly inhibited the galectin-3-induced angiogenesis. Furthermore, galectin-3 promoted the clustering of integrin alphavbeta3 and activated focal adhesion kinase. Knockdown of GnTV, an enzyme that synthesizes high-affinity glycan ligands for galectin-3, substantially reduced: (a) complex N-glycans on alphavbeta3 integrins and (b) VEGF- and bFGF-mediated angiogenesis. Collectively, these data suggest that galectin-3 modulates VEGF- and bFGF-mediated angiogenesis by binding via its carbohydrate recognition domain, to the GnTV synthesized N-glycans of integrin alphavbeta3, and subsequently activating the signaling pathways that promote the growth of new blood vessels. These findings have broad implications for developing novel, carbohydrate-based therapeutic agents for inhibition of angiogenesis.