Several dipteran insects are vectors of parasites causing major human infectious diseases. Among these, the tsetse fly, Glossina spp., is responsible for the transmission of trypanosomes, the pathogens responsible for sleeping sickness in Africa. A better understanding of insect-parasite interactions will help establish new strategies to fight this important often fatal disease. Antimicrobial peptides (AMPs) are part of the humoral immune response in insects during bacterial, fungal and parasitic infections. Here, we studied the immune response of Glossina morsitans to bacteria and to Trypanosoma brucei brucei by analyzing the synthesis of AMPs as markers of the humoral immune response. By reversed-phase chromatography, mass spectrometry analysis, Edman degradation and in vitro antimicrobial assays of the hemolymph of immune-challenged adults of G. morsitans, we identified three AMPs: a cecropin, an attacin and a defensin. These three AMPs were found to be induced upon systemic bacterial infection and also after per os infections by bacteria and parasites.