Abstract Cancer metabolism has been well documented to influence primary tumor development. Yet, the role of deregulated metabolism in metastasis progression is not well understood. Based on a distinct metabolic transcriptome profile in brain metastases compared to their primary tumor counterparts, we hypothesized that metabolic transcriptome shifting during metastatic evolution is crucial for metastatic success to the brain. Here we show that, despite a global down-regulation of multiple metabolic pathways in the brain metastatic tumors, the expression of the GABA metabolic pathway mediator glutamate decarboxylase 1 (GAD1) is significantly up-regulated. Using cell based co-culture models for different primary and brain metastatic microenvironments and in vivo brain metastasis models, we demonstrate that down-regulation of DNA methyltransferase 1 (DNMT1) induced by the brain microenvironment results in decreased GAD1 promoter methylation and subsequent up-regulation of GAD1 expression in tumor cells. To dynamically visualize the cellular metabolic responses mediated by GAD1, we utilized the Peredox biosensor to monitor the cytosolic NADH:NAD+ equilibrium in tumor cells using time-lapse imaging. By knocking down of GAD1 induced by primary glia cell co-culture, we abolished the capability of tumor cells to utilize extracellular glutamine, leading to an NADH accumulation in the cytosol and a more oxidative cellular status. Lastly, either loss of GAD1 genetically or targeting GABA metabolic pathway by the repurposing of a neurological drug, vigabatrin, results in a significant decrease in brain metastasis incidence. Taken together, our results demonstrated that brain microenvironment-specific metabolic shifting through GAD1 promoter de-methylation drives brain metastasis outgrowth. Citation Format: Patricia M. Schnepp, Dennis D. Lee, Ian H. Guldner, Treasa O'Tighearnaigh, Bhavana Palakurthi, Kaitlyn E. Eckert, Tiffany A. Toni, Brandon L. Ashfeld, Siyuan Zhang. Brain metastatic microenvironment reshapes cancer cell metabolism through epigenetic up-regulation of glutamate decarboxylase 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4934. doi:10.1158/1538-7445.AM2017-4934