Food allergy as a global health problem threatens food industry. Bee pollen (BP) is a typical food with allergenic potentials, although it performs various nutritional/pharmacological functions to humans. In this study, lactic acid bacteria (LAB) were used to ferment Brassica napus BP for alleviating its allergenicity. Four novel allergens (glutaredoxin, oleosin-B2, catalase and lipase) were identified with significant decreases in LAB-fermented BP (FBP) than natural BP by proteomics. Meanwhile, metabolomics analysis showed significant increases of 28 characteristic oligopeptides and amino acids in FBP versus BP, indicating the degradation of LAB on allergens. Moreover, FBP showed alleviatory effects in BALB/c mice, which relieved pathological symptoms and lowered production of allergic mediators. Microbial high-throughput sequencing analysis showed that FBP could regulate gut microbiota and metabolism to strengthen immunity, which were closely correlated with the alleviation of allergic reactivity. These findings could contribute to the development and utilization of hypoallergenic BP products.