Purpose <p>Melittin, a 26-residue amphipathic peptide and the principal component of honeybee venom, exhibits broad cytolytic activity against various cancer cells. An elevated M₂/M₁ macrophage ratio in HER2-expressing breast cancer might promote tumor progression. This study investigates the cytotoxic, apoptotic and macrophage polarization effects of melittin in breast cancer cells, including MCF-7 and HER2-expressing SK-BR-3, both alone and in the presence of the macrophages.</p> Method <p>The cytotoxicity of melittin on MCF-7, SK-BR-3, THP-1 and CCD-34Lu cells was assessed using the MTT assay. The apoptotic effect of the peptide was determined via Annexin V-FITC/PI staining and flow cytometric analysis. THP-1 cells were then differentiated into M₀ macrophages by incubating with 15 ng/ml PMA (phorbol 12-myristate 13-acetate) for 48&#xa0;h and treated with melittin to evaluate its effect on macrophage polarization. Co-culture models of macrophages with MCF-7 or SK-BR-3 cells were created using transwell insert plates, and melittin’s effect on macrophage polarization and apoptosis were analyzed by flow cytometry.</p> Results <p>Melittin displayed greater cytotoxic activity in MCF-7 and SK-BR-3 cells than in non-malignant cell lines. Apoptosis assay confirmed that melittin induced apoptosis in cancer cells. Furthermore, melittin treatment promoted both M₁ and M₂ macrophages polarization, with co-culture models highlighting its ability to induce M₁ macrophage and apoptosis in both MCF-7 and SK-BR-3 cells.</p> Conclusion <p>These findings provide in vitro evidence that melittin can induce apoptosis and promote anti-tumoral macrophage polarization, suggest its potential as an immunomodulatory agent and provide supportive data for future preclinical and translational studies of HER2-expressing breast cancer.</p> Graphical Abstract <p></p>