Objective: Pyruvate kinases M (PKM), including the PKM1 and PKM2 isoforms, are critical factors in glucose metabolism. PKM2 promotes aerobic glycolysis, a phenomenon known as the Warburg effect. The purpose of this study was to identify the roles of PKM2 in regulating cellular metabolism. Methods: The CRISPR/Cas9 system was used to generate the PKM-knockout cell model to evaluate the role of PKM in cellular metabolism. Lactate levels were measured by the Vitros LAC slide method on an autoanalyzer and glucose levels were measured by the autoanalyzer AU5800. The metabolism of 13C6-glucose or 13C5-glutamine was evaluated by liquid chromatography/mass spectrometry analyses. The effects of PKM on tumor growth were detected in vivo in a tumor-bearing mouse model. Results: We found that both PKM1 and PKM2 enabled aerobic glycolysis, but PKM2 converted glucose to lactate much more efficiently than PKM1. As a result, PKM2 reduced glucose levels reserved for intracellular utilization, particularly for the production of citrate, and thus increased the α-ketoglutarate/citrate ratio to promote the generation of glutamine-derived acetylcoenzyme A through the reductive pathway. Furthermore, reductive glutamine metabolism facilitated cell proliferation under hypoxia conditions, which supports in vivo tumor growth. In addition, PKM-deletion induced a reverse Warburg effect in tumorassociated stromal cells. Conclusions: PKM2 plays a critical role in promoting reductive glutamine metabolism and maintaining proton homeostasis. This study is helpful to increase the understanding of the physiological role of PKM2 in cancer cells.