In humans, complex I of the respiratory chain is composed of seven mitochondrial DNA (mtDNA)-encoded and 38 nuclear-encoded subunits that assemble together in a process that is poorly defined. To date, only two complex I assembly factors have been identified and how each functions is not clear. Here, we show that the human complex I assembly factor CIA30 (complex I intermediate associated protein) associates with newly translated mtDNA-encoded complex I subunits at early stages in their assembly before dissociating at a later stage. Using antibodies we identified a CIA30-deficient patient who presented with cardioencephalomyopathy and reduced levels and activity of complex I. Genetic analysis revealed the patient had mutations in both alleles of the NDUFAF1 gene that encodes CIA30. Complex I assembly in patient cells was defective at early stages with subunits being degraded. Complementing the deficiency in patient fibroblasts with normal CIA30 using a novel lentiviral system restored steady-state complex I levels. Our results indicate that CIA30 is a crucial component in the early assembly of complex I and mutations in its gene can cause mitochondrial disease.