Abstract Plants used in Ayurvedic medicine, which has been practiced for centuries in India for the treatment of different ailments, continue to draw attention for identification of novel small molecule cancer chemopreventive agents. Withaferin A (WA) is one such promising small molecule constituent of the medicinal plant Withania somnifera (also known as Ashwagandha) with in vivo activity against xenografted human breast cancer cells. We have also shown recently that mammary cancer development and pulmonary metastasis in MMTV-neu transgenic mice is inhibited significantly by WA administration. In the present study, we performed metabolomics profiling using plasma and tumor tissues (n=8) and proteomics profiling using tumor tissues (n=3) from control and WA-treated MMTV-neu mice to gain insights into the mechanism underlying in vivo preventive effect of WA. A total of 320 and 328 biochemicals of known identity were detectable in the plasma and tumor tissue, respectively. The WA-mediated mammary cancer prevention in MMTV-neu mice was associated with a statistically significant alteration (P≤0.05) in the levels of 76 biochemicals in the plasma (24 increase, 52 decrease). Metabolomics using tumor tissues revealed alterations in the levels of 24 biochemicals (2 increase, 24 decrease) upon WA administration. One change that was highly consistent in the plasma and tumor tissue from WA-treated mice compared to controls was a decrease in glucose utilization, glycolysis, and TCA cycle activity. Proteomics using tumor tissues confirmed downregulation of many glycolysis-related proteins in the tumor of WA-treated mice compared with control, including M2-type pyruvate kinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-bisphosphate aldolase A isoform 2. Downregulation of TCA cycle-related proteins in the tumor of WA-treated mice compared with control included pyruvate carboxylase and isocitrate dehydrogenase. Other altered biochemicals were reflective of changes in arginine metabolism, polyamine biosynthesis, glutathione metabolism, lipid metabolism, and catabolism of branched-chain amino acids. These observations indicate that WA treatment prevents mammary cancer in MMTV-neu mice by suppressing glycolysis (Warburg effect) and TCA cycle. This study was supported by the grant CA142604 awarded by the National Cancer Institute. Citation Format: Shivendra V. Singh, Eun-Ryeong Hahm, Joomin Lee, Su-Hyeong Kim, Anuradha Sehrawat, Julie A. Arlotti. Metabolomics and proteomics reveal inhibition of glycolysis in withaferin A-mediated prevention of mammary carcinogenesis in MMTV-neu mice. [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 3714. doi:10.1158/1538-7445.AM2013-3714