Platelet activation is a critical process during inflammation, thrombosis, and cancer. Here, we show that galectin-1, an endogenous lectin with immunoregulatory properties, plays a key role in human platelet activation and function. Galectin-1 binds to human platelets in a carbohydrate-dependent manner and synergizes with ADP or thrombin to induce platelet aggregation and ATP release. Furthermore, galectin-1 induces F-actin polymerization, up-regulation of P-selectin, and GPIIIa expression; promotes shedding of microvesicles; and triggers conformational changes in GPIIb/IIIa. In addition, exposure to this lectin favors the generation of leukocyte-platelet aggregates. A further mechanistic analysis revealed the involvement of Ca²⁺ and cyclic nucleotide-dependent pathways in galectin-1-mediated control of platelet activation. Finally, expression of endogenous galectin-1 in human platelets contributes to ADP-induced aggregation. Our study reveals a novel unrecognized role for galectin-1 in the control of platelet physiology with potential implications in thrombosis, inflammation, and metastasis.—Pacienza, N., Pozner, R. G., Bianco, G. A., D'Atri, L. P., Croci, D. O., Negrotto, S., Malaver, E., Gómez, R. M., Rabinovich, G. A., Schattner, M. The immunoregulatory glycan-binding protein galectin-1 triggers human platelet activation.