We generated mice deficient in Lon protease (LONP1), a major enzyme of the mitochondrial quality control machinery. Homozygous deletion of Lonp1 causes early embryonic lethality, whereas its haploinsufficiency protects against colorectal and skin tumors. Furthermore, LONP1 knockdown inhibits cellular proliferation and tumor and metastasis formation, whereas its overexpression increases tumorigenesis. Clinical studies indicate that high levels of LONP1 are a poor prognosis marker in human colorectal cancer and melanoma. Additionally, functional analyses show that LONP1 plays a key role in metabolic reprogramming by remodeling OXPHOS complexes and protecting against senescence. Our findings demonstrate the relevance of LONP1 for cellular and organismal viability and identify this protease as a central regulator of mitochondrial activity in oncogenesis. Display omitted •LONP1 is a mitochondrial protease essential for embryonic development•LONP1 is upregulated in human colorectal and melanoma tumors•Loss of LONP1 induces mitochondrial dysfunction and triggers cellular senescence•LONP1 regulates metabolic reprogramming through remodeling mitochondrial function In this study, Quiros et al. identify LONP1 as an essential gatekeeper protease for ensuring cellular and organismal fitness. LONP1 deficiency causes embryonic lethality in mice, and haploinsufficiency protects against colorectal and skin tumors. LONP1 participates in OXPHOS remodeling and allows efficient mitochondrial and cellular reprogramming, thus facilitating the tumorigenic properties of cancer cells. This study provides essential functional knowledge about LONP1 and suggests new opportunities for therapeutic intervention in cancer.