The two prohibitins (PHBs) family member proteins, PHB1 and PHB2, are localized in the mitochondria and the nucleus of eukaryotic organisms, and are associated with a number of conserved functions. Several reports of conflict exist about the exact roles these proteins are playing in the cell biology of both metazoa and unicellular organisms, and whether these proteins are mainly involved in mitochondrial-related functions or nuclear related functions. In this study, we used the model system of Trypanosoma brucei in tracing expression, localization and cellular function of one of the two PHB proteins, TbPHB1. TbPHB1 showed high degree of homology to members of PHB family proteins in other eukaryotes. When expressed as cMyc-tagged recombinant protein, TbPHB1 was localized mainly to the single mitochondrion of T. brucei cell as revealed by its co- localization with the specific mitochondrial marker of Mitotracker. Balanced physiological expression of TbPHB1 is very essential as revealed by the growth inhibition and lethal phenotype after overexpressing the protein for an extended period of time. Most importantly, microscopy analysis of cells with overexpressed TbPHB1 revealed an interesting phenotype in which the mitochondria of affected cells showed severe distortion in the form of two fragmented bodies at two ends, rather than the homogeneous staining throughout cell vicinity that observed in cells with biologically normal expression of PHB1. Taken together, data demonstrate a functional role of PHB1 in T. brucei cells, and namely, in the maintenance of mitochondrial integrity as demonstrated by its mitochondrial localization and the mitochondria distortion phenotype after the protein overexpression.