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Tsetse flies (Glossina spp.) are major vectors of African trypanosomes, causing either Human or Animal African Trypanosomiasis (HAT or AAT). Several approaches have been developed to control the disease, among which is the anti‐vector Sterile Insect Technique. Another approach to anti‐vector strategies could consist of controlling the fly's vector competence through hitherto unidentified regulatory factors (genes, proteins, biological pathways, etc.). The present work aims to evaluate the protein abundance in the midgut of wild tsetse flies (Glossina palpalis palpalis) naturally infected by Trypanosoma congolense s.l. Infected and non‐infected flies were sampled in two HAT/AAT foci in Southern Cameroon. After dissection, the proteomes from the guts of parasite‐infected flies were compared to that of uninfected flies to identify quantitative and/or qualitative changes associated with infection. Among the proteins with increased abundance were fructose‐1,6‐biphosphatase, membrane trafficking proteins, death proteins (or apoptosis proteins) and SERPINs (inhibitor of serine proteases, enzymes considered as trypanosome virulence factors) that displayed the highest increased abundance. The present study, together with previous proteomic and transcriptomic studies on the secretome of trypanosomes from tsetse fly gut extracts, provides data to be explored in further investigations on, for example, mammal host immunisation or on fly vector competence modification via para‐transgenic approaches. [ABSTRACT FROM AUTHOR]