To unravel the genotype–phenotype relationship and underlying pathogenic mechanism between OXA1L variants and mitochondrial diseases, patient‐specific human‐induced pluripotent stem cells (hiPSC) were reprogrammed and differentiated into myotubes, while OXA1L knockout human immortalised skeletal muscle cells (IHSMC) and a conditional skeletal muscle knockout mouse model was generated using clustered regularly interspaced short palindromic repeats/Cas9 genomic editing technology. Here, OXA1L is linked to mitochondrial disease.