Platelets are multifunctional cellular mediators in many physiological and pathophysiological processes such as thrombosis, angiogenesis, and inflammation. Several members of galectins, a family of carbohydrate-binding proteins with a broad range of immunomodulatory actions, have been reported to activate platelets.In this study, we investigated the role of galectin-9 (Gal-9) as a novel ligand for platelet glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2).Platelet spreading, aggregation, and P-selectin expression in response to Gal-9 were measured in washed platelet suspensions via static adhesion assay, light transmission aggregometry, and flow cytometry, respectively. Solid-phase binding assay and protein phosphorylation studies were utilized to validate the interaction between Gal-9 and GPVI, and immunoprecipitation for detecting CLEC-2 phosphorylation. Wild-type (WT), GPVI-knockout (Gp6-/- ), and GPVI and CLEC-2-double knockout (Gp6-/- /Gp1ba-Cre-Clec1bfl/fl ) mice were used.We have shown that recombinant Gal-9 stimulates aggregation in human and mouse washed platelets dose-dependently. Platelets from both species adhere and spread on immobilized Gal-9 and express P-selectin. Gal-9 competitively inhibited the binding of human recombinant D1 and D2 domains of GPVI to collagen. Gal-9 stimulated tyrosine phosphorylation of CLEC-2 and proteins known to lie downstream of GPVI and CLEC-2 including spleen tyrosine kinase and linker of activated T cells in human platelets. GPVI-deficient murine platelets exhibited significantly impaired aggregation in response to Gal-9, which was further abrogated in GPVI and CLEC-2-double-deficient platelets.We have identified Gal-9 as a novel platelet agonist that induces activation through interaction with GPVI and CLEC-2.