Traditional thermal processing of cashews not only results in nutrient loss and harmful by-products, but also does not significantly reduce allergenicity. Irradiation could be an important non-thermal processing method to reduce cashew allergens’ allergenicity and retain their nutritional properties. This study aimed to evaluate the effects of gamma irradiation processing on the structure and potential allergenicity of Ana o 3. The Ana o 3 solutions were gamma-irradiated at 0, 1, 3, 5, and 10 kGy. The structure change was monitored by Tricine-SDS-PAGE, circular dichroism spectroscopy, and fluorescence spectroscopy. The potential allergenicity was tested by immunoblotting, indirect competitive ELISA, and the human basophil KU812 degranulation assay using serum from cashew allergy patients. The results of CD spectroscopy showed that the content of α-helices decreased from 46.8% to 30.9% after 3 to 10 kGy, while the content of random coils increased from 23.7% to 33.3%. Meanwhile, a large number of hydrophobic regions were exposed, resulting in an increase in the hydrophobic surface of the protein. In terms of allergenicity, the IC₅₀ values obtained by the competitive inhibition ELISA after irradiation increased from 0.628 to 4.054 μg mL⁻¹, indicating that irradiation reduced the IgE binding capacity of Ana o 3, which was consistent with the results of western blotting. In addition, the basophil degranulation analysis showed that the release of IL-6, TNF-α, and histamine was decreased. It was shown that the potential allergenicity of the irradiated Ana o 3 was remarkably decreased since irradiation could mask or destroy the allergen epitopes, providing a new approach to reduce the allergenicity of cashew products.