Background:  Systemic side‐effects of venom immunotherapy (VIT) represent a considerable problem in the treatment of patients allergic to Hymenoptera venom. We examined the hypothesis whether basophil responsiveness might be connected with the adverse reactions to VIT. Methods:  Basophil surface expression of activation marker CD63 induced by different concentrations of honeybee and wasp venom (0.1 and 1 μg/ml) was measured by flow cytometry in 34 patients with history of systemic anaphylactic reactions to Hymenoptera sting just before rush honeybee or wasp VIT. Results:  Eleven of 34 patients had systemic anaphylactic reaction (Mueller grades I–III) and one patient a large local reaction to VIT. In those 12 patients, median percentage of activated basophils after stimulation with VIT‐specific venom in concentration of 0.1 μg/ml was 99% (range: 17–195) of value reached with stimulation with 1 μg/ml. Side‐effects occurred in all patients with 0.1/1 ratios over 92% (eight of 12). In contrast, in 22 patients with no side‐effects, the median 0.1/1 ratio was 25% (range: 2–92). These concentration‐dependent activation ratios were significantly different between the groups with and without side reactions (P < 0.0001). We also show significant positive correlation of the occurrence/clinical grade of the side‐effects with individual ratios of CD63 basophil response (r = 0.73, P < 0.0001). Conclusion:  The results suggest that increased basophil sensitivity to allergen‐specific in vitro stimulation is significantly associated with major side‐effects of VIT.