Pachymic acid (PA), a triterpenoid from Poria cocos, has various pharmacological effects, including anti-inflammatory, anti-cancer, anti-aging, and insulin-like properties. PA has gained considerable research attention, but the mechanism of its anti-cancer effects remains unclear. In this study, pyruvate kinase M2 (PKM2) was discovered as a PA target via the drug affinity responsive target stability. Molecular docking and enzyme assay revealed that PA is a competing activator of PKM2, and mimics the natural activator, fructose-1,6-bisphosphate. PKM2 activation should augment the flux of glycolysis. However, decreased glucose uptake and lactate production after PA treatment was observed in SK-BR-3 breast carcinoma cells, indicating a blockage or downregulation of glycolysis. The potential of previously reported triterpenoids in blocking hexokinase II (HK2) activity inspired us to investigate the inhibition effect of PA on HK2 activity. Molecular docking and enzyme assay confirmed that PA was an inhibitor of HK2, with an IC₅₀ of 5.01 µM. The possible consequences of glycometabolic regulation by PA, such as dissociation of HK2 from the mitochondria, release of mitochondrial cytochrome (Cyt) c, depletion of ATP, and generation of reactive oxygen species, were further validated. Furthermore, the details of the possible linkage of targeting PKM2 and HK2 with previously reported actions of PA were discussed. The results of our study provided valuable information on the anti-cancer mechanisms of PA.