Abstract Yeast Coq5p is required for the biosynthesis of coenzyme Q6 (CoQ6), but its human homolog has not been studied. We purified soluble recombinant human COQ5 protein under native conditions and generated an antibody recognizing both precursor and mature forms of COQ5. Mitochondrial localization of the mature form in 143B cells was demonstrated with this antibody. Moreover, a chemical uncoupler in a dose that suppressed CoQ10 levels downregulated the mature form but augmented the precursor form of COQ5. The results that knockdown of the COQ5 gene reduced CoQ10 levels further indicated the critical role of COQ5 in the biosynthesis of CoQ10.