Insects, such as Drosophila melanogaster, rely on innate immune defences to combat microbial threats. Antimicrobial peptides (AMPs) play an important role in limiting pathogen entry and colonization. Despite intensive research into the regulation and biochemical properties of antimicrobial peptides, their exact significance in vivo has remained uncertain due to the challenges of mutating small genes. Fortunately, recent technologies have enabled the mutation of individual antimicrobial peptide genes, to overcome previous obstacles and opened new avenues for research. In this study, we characterized one novel host-defence peptide, Paillotin (IM18, CG33706), using loss-of-function mutants. Paillotin is an ancient host-defence peptide of Diptera, regulated by the Immune deficiency (Imd) pathway. Loss of Paillotin does not impact the activity of either the Imd or Toll pathways. Importantly, we found that Paillotin mutants are viable but exhibit increased susceptibility to specific infections, particularly Providencia burhodogranariea. Paillotin was further found to contribute synergistically to defence against P. burhodogranariea when combined with other antimicrobial peptides. However, we did not detect direct microbicidal activity of Paillotin in vitro in our study. Taken together, our findings identify Paillotin as a novel host-defence peptide acting downstream of Imd signalling, advancing our understanding of the Drosophila antimicrobial response.