In mice and humans with cancer, intravenous ¹³ C-glucose infusion results in ¹³ C labeling of tumor tricarboxylic acid (TCA) cycle intermediates, indicating that pyruvate oxidation in the TCA cycle occurs in tumors. The TCA cycle is usually coupled to the electron transport chain (ETC) because NADH generated by the cycle is reoxidized to NAD ⁺ by the ETC. However, ¹³ C labeling does not directly report ETC activity, and other pathways can oxidize NADH, so the ETC’s role in these labeling patterns is unverified. We examined the impact of the ETC complex I inhibitor IACS-010759 on tumor ¹³ C labeling. IACS-010759 suppresses TCA cycle labeling from glucose or lactate and increases labeling from glutamine. Cancer cells expressing yeast NADH dehydrogenase-1, which recycles NADH to NAD ⁺ independently of complex I, display normalized labeling when complex I is inhibited, indicating that cancer cell ETC activity regulates TCA cycle metabolism and ¹³ C labeling from multiple nutrients.