Abstract Bee venom (apitoxin) is a mixture of bioactive molecules, with melittin as its principal component. Although its therapeutic potential is increasingly recognized, its toxic effects on vascular homeostasis remain underexplored. We investigated the impact of apitoxin and melittin on vascular cell viability and mouse aortic function. Cytotoxicity was assessed in cultured endothelial and smooth muscle cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Aortic function was evaluated by mounting thoracic aortas from young male and female C57BL/6J mice in tissue baths. Isometric tension was measured during phenylephrine-induced contractions, as well as endothelium-dependent (acetylcholine) and -independent (sodium nitroprusside) relaxations. To evaluate the roles of nitric oxide (NO) and oxidative stress, we used the NO synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) and the antioxidant superoxide dismutase (SOD), respectively. High-performance liquid chromatography analysis revealed that melittin comprised 43.80% of apitoxin. Both apitoxin and melittin exhibited concentration-dependent cytotoxicity, significantly reducing endothelial cell viability at concentrations ≥5 µg/ml, whereas smooth muscle cells were affected at lower concentrations (≥2.5 µg/ml for apitoxin; ≥1.5 µg/ml for melittin). In functional experiments, apitoxin enhanced phenylephrine-induced contractions at 1 µg/ml and impaired both endothelium-dependent and -independent relaxations at ≥0.1 µg/ml, particularly in males. Although melittin mimicked these effects, higher concentrations (≥5 µg/ml) were required, suggesting that other venom components contribute to the vascular functional toxicity of apitoxin. L-NAME and SOD prevented apitoxin-induced vascular impairments, implicating the NO pathway and oxidative stress. These findings demonstrate that apitoxin impairs vascular cell viability and aortic function at clinically relevant concentrations, underscoring both its vascular risks and therapeutic potential.