Reductive glutamine metabolism via IDH1 mediates lipogenesis under hypoxia. In mammalian cells, acetyl coenzyme A (AcCoA) is a key precursor for fatty acid synthesis and protein acetylation. Normally, AcCoA is generated from glucose-derived pyruvate through the citrate shuttle and adenosine triphosphate citrate lyase (ACL) in the cytosol. However, under hypoxia, proliferating cells shift to aerobic glycolysis, diverting glucose carbon away from the tricarboxylic acid (TCA) cycle and fatty acid synthesis. Although glutamine is consumed in excess of nitrogen biosynthesis needs, its metabolism in hypoxic cells remains poorly understood. This study shows that human cells use reductive metabolism of alpha-ketoglutarate (αKG) to generate AcCoA for lipid synthesis. This isocitrate dehydrogenase 1 (IDH1)-dependent pathway is active under normal conditions but cells under hypoxia rely almost exclusively on reductive carboxylation of glutamine-derived αKG for de novo lipogenesis. Renal cell lines deficient in the von Hippel-Lindau (VHL) tumor suppressor protein preferentially use reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results highlight the critical role of oxygen in regulating carbon utilization to produce AcCoA and support lipid synthesis in mammalian cells. Hypoxic cells exhibit a shift toward aerobic glycolysis, but a functional electron transport chain and glutamine-derived carbon are required for most transformed cells. The study observed increased glucose consumption and lactate secretion in A549 cells cultured at ~1% oxygen. Glutamine consumption also increased while glutamate secretion remained unchanged, indicating elevated net glutamine consumption and suggesting that glutamine carbon is used for biosynthesis. Glutamine can contribute carbon to lipogenic AcCoA through two pathways: oxidative metabolism in the TCA cycle or reductive carboxylation to generate citrate. To determine which pathway cells use, stable isotopic tracers were used. The study found that reductive carboxylation contributes significantly to the cytosolic AcCoA pool. Quantifying the specific contributions of oxidative and reductive glutamine metabolism to fatty acid synthesis, the study used tracers and isotopomer spectral analysis. The results showed that reductive carboxylation is the primary route for glutamine, glutamate, and αKG carbon to be converted to lipids in cultured cells. The study also found that IDH1 is involved in reductive carboxylation, and its knockdown impaired cell proliferation. Hypoxia increased reductive carboxylation activity, and under hypoxia, fatty acids were primarily synthesized from glutamine carbon via the reductive pathway. The study also found that hypoxia increases the dependence of cells on glutamine, as evidenced by decreased proliferation in the absenceReductive glutamine metabolism via IDH1 mediates lipogenesis under hypoxia. In mammalian cells, acetyl coenzyme A (AcCoA) is a key precursor for fatty acid synthesis and protein acetylation. Normally, AcCoA is generated from glucose-derived pyruvate through the citrate shuttle and adenosine triphosphate citrate lyase (ACL) in the cytosol. However, under hypoxia, proliferating cells shift to aerobic glycolysis, diverting glucose carbon away from the tricarboxylic acid (TCA) cycle and fatty acid synthesis. Although glutamine is consumed in excess of nitrogen biosynthesis needs, its metabolism in hypoxic cells remains poorly understood. This study shows that human cells use reductive metabolism of alpha-ketoglutarate (αKG) to generate AcCoA for lipid synthesis. This isocitrate dehydrogenase 1 (IDH1)-dependent pathway is active under normal conditions but cells under hypoxia rely almost exclusively on reductive carboxylation of glutamine-derived αKG for de novo lipogenesis. Renal cell lines deficient in the von Hippel-Lindau (VHL) tumor suppressor protein preferentially use reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results highlight the critical role of oxygen in regulating carbon utilization to produce AcCoA and support lipid synthesis in mammalian cells. Hypoxic cells exhibit a shift toward aerobic glycolysis, but a functional electron transport chain and glutamine-derived carbon are required for most transformed cells. The study observed increased glucose consumption and lactate secretion in A549 cells cultured at ~1% oxygen. Glutamine consumption also increased while glutamate secretion remained unchanged, indicating elevated net glutamine consumption and suggesting that glutamine carbon is used for biosynthesis. Glutamine can contribute carbon to lipogenic AcCoA through two pathways: oxidative metabolism in the TCA cycle or reductive carboxylation to generate citrate. To determine which pathway cells use, stable isotopic tracers were used. The study found that reductive carboxylation contributes significantly to the cytosolic AcCoA pool. Quantifying the specific contributions of oxidative and reductive glutamine metabolism to fatty acid synthesis, the study used tracers and isotopomer spectral analysis. The results showed that reductive carboxylation is the primary route for glutamine, glutamate, and αKG carbon to be converted to lipids in cultured cells. The study also found that IDH1 is involved in reductive carboxylation, and its knockdown impaired cell proliferation. Hypoxia increased reductive carboxylation activity, and under hypoxia, fatty acids were primarily synthesized from glutamine carbon via the reductive pathway. The study also found that hypoxia increases the dependence of cells on glutamine, as evidenced by decreased proliferation in the absence