This study aimed to investigate the venom sac extracts (VSE) of the European hornet (EH) Vespa crabro (Linnaeus, 1758) (Hymenoptera: Vespidae), focusing on the differences between stinging females, gynes (G) and workers (W), at the protein level. Using a quantitative “Sequential Window Acquisition of all Theoretical Fragment Ion Mass Spectra” (SWATH-MS) analysis, we identified and quantified a total of 240 proteins. Notably, within the group, 45.8 % (n = 110) showed significant differential expression between VSE-G and VSE-W. In this set, 57.3 % (n = 63) were upregulated and 42.7 % (n = 47) downregulated in the G. Additionally, the 200 quantified proteins from the class Insecta belong to 16 different species, six of them to he Hymenoptera/Apidae lineage, comprising seven proteins with known potential as allergens. Phospholipase A1 (Vesp v 1), phospholipase A1 verutoxin 2b (VT-2b), hyaluronidase A (Vesp v 2A), hyaluronidase B (Vesp v 2B), and venom allergen 5 (Vesp v 5) were significantly downregulated in the G, and vitellogenin (Ves v 6) was upregulated. Overall, 46 % of the VSE proteins showed differential expression, with a majority being upregulated in G. These findings shed light on the proteomic differences in VSE between EH castes, potentially contributing to our understanding of their behavior and offering insights for allergy research.