BACKGROUND Horsefly sting causes allergic reactions in human body. However, our knowledge on horsefly allergens remains poor. OBJECTIVES To identify the novel horsefly allergens and characterize their properties. METHODS A native allergen protein Tab y 1 (apyrase) was purified from the salivary glands of the horsefly Tabanus yao Macquart by gel filtration and ion exchange chromatography. Its sequence was determined by Edman degradation and cDNA cloning. Its allergenicity was assessed by immunoblotting for specific IgE, basophil activation test, skin prick test (SPT), and competitive enzyme-linked immunosorbent assay (ELISA). RESULTS Tab y 1 showed a single diffusion band of 70 kDa on SDS-PAGE. Seventy percent (7/10) of patients with horsefly allergy tested positive to Tab y 1 in SPT; sera from 81% (30/37) of patients reacted to Tab y 1 on western blots. Purified Tab y 1 reduced approximately 42% sera IgE reactivity to horsefly salivary gland extract on a competitive ELISA. Tab y 1 upregulated the expression of CD63 and CCR3 on passively sensitized basophils by up to approximately 4.9-fold. Tab y 1 also showed enzymatic activity to hydrolyze ATP and ADP, and potent antiplatelet aggregation and antithrombotic activities. CONCLUSION The current work identified a novel major allergen of horsefly, Tab y 1, with antiplatelet aggregation and antithrombotic activities, which implicates Tab y 1 in helping horseflies suck host blood, meanwhile causing allergy in their human hosts.