Phosphorylation of respiratory chain components has emerged as a mode of regulation of mitochondrial energy metabolism, but its mechanisms are still largely unexplored. A recently discovered intramitochondrial signalling pathway links CO₂ generated by the Krebs cycle with the respiratory chain, through the action of a mitochondrial soluble adenylyl cyclase (mt‐sAC). Cytochrome oxidase (COX), whose deficiency causes a number of fatal metabolic disorders, is a key mitochondrial enzyme activated by mt‐sAC. We have now discovered that the mt‐sAC pathway modulates mitochondrial biogenesis in a reactive oxygen species dependent manner, in cultured cells and in animals with COX deficiency. We show that upregulation of mt‐sAC normalizes reactive oxygen species production and mitochondrial biogenesis, thereby restoring mitochondrial function. This is the first example of manipulation of a mitochondrial signalling pathway to achieve a direct positive modulation of COX, with clear implications for the development of novel approaches to treat mitochondrial diseases.