Many genes inherited from the alpha-proteobacterial ancestor of mitochondria have undergone evolutionary transfer to the nuclear genome in eukaryotes. In some rare cases, genes have been functionally transferred in pieces, resulting in split proteins that presumably interact in trans within mitochondria, fulfilling the same role as the ancestral, intact protein. We describe a nucleus-encoded mitochondrial protein (here named Cox1-c) in the amoeboid protist Acanthamoeba castellanii that is homologous to the C-terminal portion of conventional mitochondrial Cox1, whereas the corresponding portion of the mitochondrion-encoded A. castellanii Cox1 is absent. Bioinformatics searches retrieved nucleus-encoded Cox1-c homologs in most major eukaryotic supergroups; in these cases, also, the mitochondrion-encoded Cox1 lacks the corresponding C-terminal motif. These data constitute the first report of functional relocation of a portion of cox1 to the nucleus. This transfer event was likely ancient, with the resulting nuclear cox1-c being differentially activated across the eukaryotic domain.