Hemoglobin from Hermetia illucens exerts antibacterial activity by targeting bacterial membranes.
Drug-resistant bacteria pose an escalating threat to global public health, underscoring the urgent need for novel antibacterial agents. Natural green control strategies offer sustainable solutions against environmental pathogenic bacterial infections. Among them, natural antibacterial molecules derived from eukaryotic sources are undoubtedly the most compelling, owing to their safety, biocompatibility and resistance to drug resistance. The black soldier fly (Hermetia illucens, BSF) is recognized as a valuable reservoir of antibacterial factors due to its exceptional resistance to dense microbial environments. In this study, three previously uncharacterized antibacterial proteins were systematically identified from black soldier fly larvae (BSFL) using high-performance liquid chromatography-mass spectrometry (HPLC-MS). Hemoglobin (Hb) exhibited the strongest antibacterial activity in vitro, with low cytotoxicity, thermal stability, and acid resistance. Mechanistic analyses indicate that Hb compromises bacterial cell membrane integrity via biofilm disruption and direct membrane interaction, leading to nucleic acid and protein leakage, increased intracellular reactive oxygen species (ROS), and impairment of ATP-dependent energy metabolism. Furthermore, HiHb was structurally predicted to target intracellular components, including DNA topoisomerase and ribosomal proteins. Together, these multi-target effects accelerate bacterial cell death. Notably, ectopic expression of Hb markedly enhanced antibacterial ability in silkworms, suggesting that its antibacterial function is transferable across species. These findings highlight Hb derived from BSFL as a potent antibacterial protein, offering a promising resource for the development of novel therapeutics against bacterial infections and reduce the application of harmful bactericide.