Abstract The rationale of this cell based study was to examine the metabolic effects of a synthetic monocarboxylate transporter 1 (MCT1) inhibitor (AR-C155858) on multiple cellular metabolites in colorectal tumor cells using [1,2,3,4,5,6-13C6]-D-glucose or [1,2,3,4,5-13C5]-D-glutamine as the single precursor metabolic tracer. DiFi colorectal cancer cells were cultured for 24 hrs in the presence of 0.1% DMSO (vehicle control) or 100 nM of MCT-1 inhibitor. Intracellular glycolysis intermediates measured by liquid chromatography/mass spectrometry (LC/MS) included phosphoenolpyruvate (PEP), phosphoglycerate (PG), fructose 1,6 bisphosphate (FBP), Glucose-6-phosphate (G6P), Gluconate-6-phosphate, glyceraldehydes 3-phosphate (G3P), ribose-5-phosphate (R5P), seduheptulose-7-phosphate (S7P), pyruvate, and lactate. TCA cycle intermediates measured included malate, α-ketoglutarate, succinate, fumarate, and citrate. Both glutamine and glutamate were also measured. Increases in PEP, PG3, G6P, Gluconate-6-phosphate, G3P, S7P, pyruvate, and lactate were induced by MCT1 inhibitor when traced with 13C6 glucose. When traced with 13C5 glutamine, PEP, G6P, S7P, pyruvate, and lactate were increased by MCT1 inhibitor. In contrast, decreases in α-ketoglutarate and glutamate were seen with both tracers. Citation Format: Bo Tan, Ming-Shang Kuo, Matthew D. Breyer, Ling Liu, Yang Zhao. Stable isotope resolved metabolomics of colorectal cancer cells treated with inhibitor of monocarboxylate transporter-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1866. doi:10.1158/1538-7445.AM2013-1866