Hypoxia-inducible factor-1 (HIF-1) plays a critical role in reprogramming cancer metabolism toward aerobic glycolysis (i.e., the Warburg effect), which is critical to supplying cancer cells with the biomass needed for proliferation. Previous studies have shown that cetuximab, an EGF receptor–blocking monoclonal antibody, downregulates the alpha subunit of HIF-1 (HIF-1α) through the inhibition of EGF receptor downstream cell signaling and that downregulation of HIF-1α is required for cetuximab-induced antiproliferative effects. However, the mechanism underlying these actions has yet to be identified. In this study, we used the Seahorse XF96 extracellular flux analyzer to assess the effect of cetuximab treatment on changes in glycolysis and mitochondrial respiration, the two major energy-producing pathways, in live cells. We found that cetuximab downregulated lactate dehydrogenase A (LDH-A) and inhibited glycolysis in cetuximab-sensitive head and neck squamous cell carcinoma (HNSCC) cells in an HIF-1α downregulation–dependent manner. HNSCC cells with acquired cetuximab resistance expressed a high level of HIF-1α and were highly glycolytic. Overexpression of a HIF-1α mutant (HIF-1α/ΔODD) conferred resistance to cetuximab-induced G₁ phase cell-cycle arrest, which could be overcome by knockdown of LDH-A expression. Inhibition of LDH-A activity with oxamate enhanced the response of cetuximab-resistant cells to cetuximab. Cetuximab had no noticeable inhibitory effect on glycolysis in nontransformed cells. These findings provide novel mechanistic insights into cetuximab-induced cell-cycle arrest from the perspective of cancer metabolism and suggest novel strategies for enhancing cetuximab response. Mol Cancer Ther; 12(10); 2187–99. ©2013 AACR.