Many cancer cells exhibit enhanced glycolysis, which is seen as one of the hallmark metabolic alterations, known as Warburg effect. Substantial evidence shows that upregulated glycolytic enzymes are often linked to malignant growth. Using glycolytic inhibitors for anticancer treatment has become appealing in recent years for therapeutic intervention in cancers with highly glycolytic characteristic, including non-small cell lung cancer (NSCLC). In this work, we studied the anticancer effects and the underlying mechanisms of combination of benzerazide hydrocholoride (Benz), a hexokinase 2 (HK2) inhibitor and 64, a pyruvate dehydrogenase kinase 1 (PDK1) inhibitor, in several NSCLC cell lines. We found that combination of Benz and 64 exhibited strong synergistic anticancer effects in NCI–H1975, HCC827, NCI–H1299 and SK-LU-1 cell lines. With this combination treatment, we observed changes of certain mechanistic determinants associated with metabolic stress caused by glycolysis restriction, such as mitochondrial membrane potential depolarization, overproduction of reactive oxygen species [1], activation of AMPK and down-regulation of mTOR, which contributed to enhanced apoptosis. Moreover, Benz and 64 together significantly suppressed the tumor growth in HCC827 cell mouse xenograft model. Taken together, our study may suggest that combined inhibition of HK2 and PDK1 using Benz and 64 could be a viable anticancer strategy for NSCLC.