Coenzyme Q₁₀ (CoQ₁₀) deficiency has been associated with an increasing number of clinical phenotypes that respond to CoQ₁₀ supplementation. In two siblings with encephalomyopathy, nephropathy and severe CoQ₁₀ deficiency, a homozygous mutation was identified in the CoQ₁₀ biosynthesis gene COQ2, encoding polyprenyl-pHB transferase. To confirm the pathogenicity of this mutation, we have demonstrated that human wild-type, but not mutant COQ2, functionally complements COQ2 defective yeast. In addition, an equivalent mutation introduced in the yeast COQ2 gene also decreases both CoQ₆ concentration and growth in respiratory-chain dependent medium. Polyprenyl-pHB transferase activity was 33–45% of controls in COQ2 mutant fibroblasts. CoQ-dependent mitochondrial complexes activities were restored in deficient fibroblasts by CoQ₁₀ supplementation, and growth rate was restored in these cells by either CoQ₁₀ or uridine supplementation. This work is the first direct demonstration of the pathogenicity of a COQ2 mutation involved in human disease, and establishes yeast as a useful model to study human CoQ₁₀ deficiency. Moreover, we demonstrate that CoQ₁₀ deficiency in addition to the bioenergetics defect also impairs de novo pyrimidine synthesis, which may contribute to the pathogenesis of the disease.