The oxidative phosphorylation (OXPHOS) system in mammalian mitochondria plays a key role in harvesting energy from different types of ingested nutrients1,2. Mitochondrial metabolism is very dynamic and can be reprogrammed to support both catabolic and anabolic reactions, depending on physiological demands or disease states3. Rewiring of mitochondrial metabolism is intricately linked to common metabolic diseases4–6 and is also necessary to promote tumor growth7–11. Here, we demonstrate that per oral treatment of mice with an inhibitor of mitochondrial transcription (IMT)11 leads to a profound inhibition of mtDNA expression in the liver, which markedly reduces complex I levels of the OXPHOS system, whereas the levels of electron transfer flavoprotein dehydrogenase (ETF-DH) and other dehydrogenases that feeds electrons into the ubiquinone (Q) pool remain unaltered or are increased. Deep pProteomics and non-targeted metabolomics analyses of liver reveal that IMT-treatment rewires the OXPHOS system to activates fatty acid oxidation in mice regardless of the diet. Remarkably, treatment of high fat diet (HFD)-fed mice with IMTs rapidly normalizes body weight, reverses hepatosteatosis and restores glucose tolerance. We thus propose a novel treatment principle of obesity and diabetes based on inhibition of mtDNA transcription to accomplish a rewiring of mitochondrial metabolism to favor fatty acid degradation.