Abstract ZapE/Afg1 is a component of the inner cell membrane of some eubacteria as well as in the inner mitochondrial membrane of eukaryotes. This protein is involved in FtsZ-dependent division of bacteria and facilitates the degradation of mitochondrion-encoded subunits of respiratory complexes. Furthermore, depletion of ZapE increases resistence to apoptosis, decreases oxidative stress tolerance, and impacts mitochondrial protein homeostasis. It is unclear whether this protein is a multifunctional protein or some observed phenotypes are a secondary consequence of respiratory chain disruption. Here, we have analyzed the functions of ZapE in Trypanosoma brucei, a parasitic protist and also an important model organism. Using a newly developed BioID2 proximity-dependent biotinylation approach, we have identified the inner mitochondrial membrane insertase Oxa1 as a direct interacting partner of ZapE, which is present in two paralogs. RNAi-mediated depletion of both ZapE paralogs affected the function of respiratory complexes I and IV. We propose that it is primarily the interaction of ZapE with Oxa1, which is required for a proper insertion of many inner mitochondrial membrane proteins, that is behind the multifaceted phenotype caused by the ablation of ZapE.