Parasitic manipulations of host behaviour are known from a wide range of host–parasite associations. However, the understanding of these phenomena is far from complete and detailed investigation of their proximate causes is needed. Many studies report behavioural modifications, such as altered feeding rates in tsetse fly (Glossina) infected with the mature transmissible stage (i.e. metacyclic) of the trypanosomes. Here, bidimensional (2D) gel electrophoresis and mass spectrometry were employed to analyse and compare the head proteome between four Glossina palpalis gambiensis categories (uninfected, refractory, mature infection, immature infection). Twenty-four protein spots specifically present or absent in the head of metacyclic-infected flies were observed. These protein spots were subsequently identified and functionally classified as glycolitic, neurotransmiter synthesis, signalling, molecular chaperone and transcriptional regulation proteins. Our results indicate altered energy metabolism in the head of metacyclic-infected tsetse flies. Some of the proteins identified, such as casein kinase 2 and jun kinase have previously been shown to play critical roles in apoptosis in insect neurones. In addition, we found two pyridoxal-dependent decarboxylases (dopa decarboxylase and alpha methyldopa hypersensitive protein), suggesting a modification of serotonin and/or dopamine in the brain of metacyclic-infected tsetse flies. Our data pave the way for future investigation of the alteration of the glossina central nervous system during infection by trypanosomes.