Background & Aims: Mitochondrial dysfunction and subsequent metabolic deregulation are commonly observed in cancers, including hepatocellular carcinoma (HCC). When mitochondrial function is impaired, reductive glutamine metabolism is a major cellular carbon source for de novo lipogenesis to support cancer cell growth. The underlying regulators of reductively metabolized glutamine in mitochondrial dysfunction are not completely understood in tumorigenesis. Methods: We systematically investigated the role of oxoglutarate dehydrogenase-like (OGDHL), one of the rate-limiting components of the key mitochondrial multi-enzyme OGDH complex (OGDHC), in the regulation of lipid metabolism in hepatoma cells and mouse xenograft models. Results: Lower expression of OGDHL was associated with advanced tumor stage, significantly worse survival and more frequent tumor recurrence in 3 independent cohorts totaling 681 postoperative HCC patients. Promoter hypermethylation and DNA copy deletion of OGDHL were independently correlated with reduced OGDHL expression in HCC specimens. Additionally, OGDHL overexpression significantly inhibited the growth of hepatoma cells in mouse xenografts, while knockdown of OGDHL promoted proliferation of hepatoma cells. Mechanistically, OGDHL downregulation upregulated the alpha-ketoglutarate (alpha KG): citrate ratio by reducing OGDHC activity, which subsequently drove reductive carboxylation of glutamine-derived alpha KG via retrograde tricarboxylic acid cycling in hepatoma cells. Notably, silencing of OGDHL activated the mTORC1 signaling pathway in an alpha KG-dependent manner, inducing transcription of enzymes with key roles in de novo lipogenesis. Meanwhile, metabolic reprogramming in OGDHL-negative hepatoma cells provided an abundant supply of NADPH and glutathione to support the cellular antioxidant system. The reduction of reductive glutamine metabolism through OGDHL overexpression or glutaminase inhibitors sensitized tumor cells to sorafenib, a molecular-targeted therapy for HCC. Conclusion: Our findings established that silencing of OGDHL contributed to HCC development and survival by regulating glutamine metabolic pathways. OGDHL is a promising prognostic biomarker and therapeutic target for HCC. Lay summary: Hepatocellular carcinoma (HCC) is one of the most prevalent tumors worldwide and is correlated with a high mortality rate. In patients with HCC, lower expression of the enzyme OGDHL is significantly associated with worse survival. Herein, we show that silencing of OGDHL induces lipogenesis and influences the chemosensitization effect of sorafenib in liver cancer cells by reprogramming glutamine metabolism. OGDHL is a promising prognostic biomarker and potential therapeutic target in OGDHL-negative liver cancer. (c) 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.