Cancer cells are reprogrammed to utilize glycolysis at high rates, which provides metabolic precursors for cell growth. Consequently, glucose levels may decrease substantially in underperfused tumor areas. Gluconeogenesis results in the generation of glucose from smaller carbon substrates such as lactate and amino acids. The key gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK), has been shown to provide metabolites for cell growth. Still, the role of gluconeogenesis in cancer is unknown. Here we show that the mitochondrial isoform of PEPCK (PCK2) is expressed and active in three lung cancer cell lines and in non-small cell lung cancer samples. PCK2 expression and activity were enhanced under low-glucose conditions. PEPCK activity was elevated threefold in lung cancer samples over normal lungs. To track the conversion of metabolites along the gluconeogenesis pathway, lung cancer cell lines were incubated with (13)C₃-lactate and label enrichment in the phosphoenolpyruvate (PEP) pool was measured. Under low glucose, all three carbons from (13)C₃-lactate appeared in the PEP pool, further supporting a conversion of lactate to pyruvate, via pyruvate carboxylase to oxaloacetate, and via PCK2 to phosphoenolpyruvate. PCK2 small interfering RNA and the pharmacological PEPCK inhibitor 3-mercaptopicolinate significantly enhanced glucose depletion-induced apoptosis in A549 and H23 cells, but not in H1299 cells. The growth of H23 multicellular spheroids was significantly reduced by 3-mercaptopicolinate. The results of this study suggest that lung cancer cells may utilize at least some steps of gluconeogenesis to overcome the detrimental metabolic situation during glucose deprivation and that in human lung cancers this pathway is activated in vivo.