Galectin-1 (Gal-1) is a β-galactoside-binding lectin that regulates endothelial cell migration, proliferation, and adhesion. However, the effect of Gal-1 on vascular permeability and the underlying mechanisms are unclear. We found that high Gal-1 expression was associated with elevated tumor vascular permeability in specimens of oral squamous cell carcinoma. Using transendothelial passage of FITC-dextran and a Miles assay, we demonstrated that Gal-1 increased vascular permeability extracellularly through its carbohydrate recognition domain. Mechanism dissection revealed that the neuropilin (NRP)-1/vascular endothelial growth factor receptor- (VEGFR)-1 complex was required for Gal-1-regulated vascular permeability. Activation of VEGFR-1 triggered activation of Akt which led to a reduction in vascular endothelial-cadherin at cell-cell junctions and resulted in cytoskeletal rearrangement. Both inhibition of Gal-1 secreted from cancer cells and administration of an anti-Gal-1 antibody in the tumor microenvironment suppressed tumor growth and vascular permeability in xenograft models. In conclusion, our results demonstrate a novel function of Gal-1 of increasing vascular permeability through the NRP-1/VEGFR1 and Akt signaling pathway and indicate that targeting Gal-1 by an anti-Gal-1 antibody is a feasible therapy for vascular hyperpermeability and cancer.