Cancer cells exhibit enhanced lactate production to satisfy biosynthetic adenosine triphosphate requirements and also supply ribose 5-phosphate (R5P) and nicotinamide adenine dinucleotide phosphate via the pentose phosphate pathway (PPP). Yet, little is known about the mechanism by which glycolytic flux is diverted to PPP to fulfill the increased demand for anabolic precursors and reducing equivalents. Here we show, using a C-13-labeling methodology quantifying glycolysis and the PPP metabolism, that hypoxic cancer cells not only increase net glycolytic flux but also activate the exchange fluxes catalyzed by aldolase and transaldolase. The increased carbon exchange in the upward direction promotes the supplementation of R5P through the nonoxidative PPP and essentially controls the anaplerosis of upper glycolytic metabolites consumed for biosynthesis. This cascade of events is regulated by glyceraldehyde 3-phosphate dehydrogenase which plays a critical role in diverting metabolites for the synthesis of nucleotide precursors and thus acts as a limiting enzyme under hypoxia. (c) 2018 American Institute of Chemical Engineers