The red flour beetleTribolium castaneumis a destructive insect pest of stored food and feed products, anda model organism for development, evolutionary biology and immunity. The insect innate immune sys-tem includes antimicrobial peptides (AMPs) with a wide spectrum of targets including viruses, bacteria,fungi and parasites. Defensins are an evolutionarily-conserved class of AMPs and a potential new sourceof antimicrobial agents. In this context, we report the antimicrobial activity, phylogenetic and structuralproperties of threeT. castaneumdefensins (Def1, Def2 and Def3) and their relevance in the immunity ofT. castaneumagainst bacterial pathogens. All three recombinant defensins showed bactericidal activityagainstMicrococcus luteusandBacillus thuringiensisserovartolworthi, but only Def1 and Def2 showed abacteriostatic effect againstStaphylococcus epidermidis. None of the defensins showed activity againstthe Gram-negative bacteriaEscherichia coliandPseudomonas entomophilaor against the yeastSaccharomyces cerevisiae. All three defensins were transcriptionally upregulated following a bacterialchallenge, suggesting a key role in the immunity ofT. castaneumagainst bacterial pathogens.Phylogenetic analysis showed that defensins fromT. castaneum, mealworms, Udo longhorn beetle andhouseflies cluster within a well-defined clade of insect defensins. We conclude thatT. castaneumdefen-sins are primarily active against Gram-positive bacteria and that other AMPs may play a more prominentrole against Gram-negative species.