Bee pollen as a nutrient-rich functional food has been considered for use as an adjuvant for chronic disease therapy. However, bee pollen can trigger food-borne allergies, causing a great concern to food safety. Our previous study demonstrated that the combined use of cellulase, pectinase and papain can hydrolyze allergens into peptides and amino acids, resulting in reduced allergenicity of bee pollen based on in vitro assays. Herein, we aimed to further explore the mechanisms behind allergenicity alleviation of enzyme-treated bee pollen through a BALB/c mouse model. Results showed that the enzyme-treated bee pollen could mitigate mice scratching frequency, ameliorate histopathological injury, decrease serum IgE level, and regulate bioamine production. Moreover, enzyme-treated bee pollen can modulate metabolic pathways and gut microbiota composition in mice, further supporting the alleviatory allergenicity of enzyme-treated bee pollen. The findings could provide a foundation for further development and utilization of hypoallergenic bee pollen products.