Trypansoma brucei is a protozoan parasite that infects humans and cattle via a tsetse fly vector. Key to parasite survival during progression through this complex life cycle is the expression of cell surface and endocytic pathway glycoproteins, modified with glycosylphosphatidylinositol (GPI) membrane anchors and/or N-linked oligosaccharides. The biosynthesis of guanosine 5′-diphospho-β-L-fucose (GDP-Fuc), the activated donor for fucose, has been shown to be essential in Trypanosoma brucei. Fucose is a common constituent of eukaryotic glycan structures, but it has been rarely found in trypanosomatid glycoconjugates. A single putative T. brucei fucosyltransferase (TbFUT1) gene was identified in the genome. Unexpectedly, TbFUT1 was localized in the mitochondrion of T. brucei, suggesting that this kinetoplastid parasite possesses unprecedented mitochondrial fucosyltransferase activity. Loss of TbFUT1 by conditional knockout (cKO) in bloodstream form (BSF) mutants results in growth arrest and the accumulation of dyskinetoplastid cells lacking mitochondrial kinetoplast DNA (kDNA) yet has no pronounced effect on mitochondrial volume or structure as assessed by electron microscopy. Loss of TbFUT1 results in loss of mitochondrial membrane potential (ΨΔm) and collapse of the monomeric and dimeric forms of the F_OF₁-ATP synthase complex, as assessed by Native PAGE and Western blot analysis. However, loss of both kDNA and TbFUT1 expression is tolerated in BSF TbFUT1 cKO parasites upon mutation of the F1-γ subunit to enable ΨΔm maintenance by F₁ activity independent of F_O. Interestingly, in vitro assays indicate that a TbFUT1 preferred substrate is the Galβ1-3GlcNAc disaccharide, indicating that further glycosyltransferases (GTs) may be active in the mitochondria of T. brucei. Indeed, the mitochondrial localisation of two putative TbGTs was confirmed by C-terminal epitope tagging, and RNAi of their respective transcripts caused growth arrest, suggesting their functions may lie upstream of TbFUT1. We aim to further investigate the function of mitochondrial glycosylation by identifying the substrate(s) of TbFUT1 and characterising the activity of the remaining, putative mitochondrial TbGTs.