BackgroundThe body and head lice of humans are conspecific, but only the body louse functions as a vector to transmit bacterial pathogens such asBartonella quintana. Both louse subspecies have only two antimicrobial peptides, defensin 1 and defensin 2. Consequently, any differences in the molecular and functional properties of these two louse subspecies may be responsible for the differential vector competence between them.MethodsTo elucidate the molecular basis of vector competence, we compared differences in the structural properties and transcription factor/microRNA binding sites of the two defensins in body and head lice. Antimicrobial activity spectra were also investigated using recombinant louse defensins expressed via baculovirus.ResultsThe full-length amino acid sequences of defensin 1 were identical in both subspecies, whereas the two amino acid residues in defensin 2 were different between the two subspecies. Recombinant louse defensins showed antimicrobial activities only against the representative Gram-positiveStaphylococcus aureusbut not against either Gram-negativeEscherichia colior the yeastCandida albicans. However, they did show considerable activity againstB. quintana, with body louse defensin 2 being significantly less potent than head louse defensin 2. Regulatory sequence analysis revealed that the gene units of both defensin 1 and defensin 2 in body lice possess decreased numbers of transcription factor-binding sites but increased numbers of microRNA binding sites, suggesting relatively lower transcription activities of body louse defensins.ConclusionsThe significantly lower antibacterial activities of defensin 2 along with the reduced probability of defensin expression in body lice likely contribute to the relaxed immune response toB. quintanaproliferation and viability, resulting in higher vector competence of body lice compared to head lice.Graphical AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s13071-023-05802-4.