Introduction:Production of different antimicrobial peptides (AMPs) is one of the insect’s prominent defense strategies, regulated mainly by Toll and immune deficiency (IMD) humoral pathways. Here we focused mainly on two AMPs ofPhlebotomus papatasi, vector ofLeishmania majorparasites, their association with the relish transcription factor and the effective participation onLeishmaniainfection.Methods and results:We further characterized the role of previously described gut-specificP. papatasidefensin (PpDef1) and identified the second defensin (PpDef2) expressed in various sand fly tissues. Using the RNAi-mediated gene silencing, we report that the silencing ofPpDef1gene or simultaneous silencing of both defensin genes (PpDef1andPpDef2) resulted in increased parasite levels in the sand fly (detectable by PCR) and higher sand fly mortality. In addition, we knocked down relish, the sole transcription factor of the IMD pathway, to evaluate the association of the IMD pathway with AMPs expression inP. papatasi. We demonstrated that the relish gene knockdown reduced the expression ofPpDef2andattacin, another AMP abundantly expressed in the sand fly body.Conclusions:Altogether, our experiments show the importance of defensins in the sand fly response towardL. majorand the role of the IMD pathway in regulating AMPs inP. papatasi.