Exogenous glutamine is an important source of energy and molecular building blocks for many tumors. There is a renewed interest in therapeutically targeting glutamine metabolism due to the recent discovery of two novel glutaminase inhibitors. To quantify the dysregulation of the glutamate-glutamine equilibrium in breast cancer, metabolomics analysis of 270 clinical breast cancer and 97 normal breast samples was carried out using gas chromatography combined with time-of-flight mass spectrometry. Positive correlation between glutamate and glutamine in normal breast tissues switched to negative correlation between glutamate and glutamine in breast cancer tissues. Compared to the level of glutamate/glutamine in normal tissues, we found 56% of the ER+ tumor tissues and 88% of the ER- tumor tissues glutamate-enriched. The glutamate/glutamine ratio (GGR) significantly correlated with ER status (p=8.0E-09) and with tumor grade (p=3.3E-05). Higher levels of GGR were associated with prolonged overall survival in univariate analysis (HR=0.77, p=0.027) and in multivariate analysis (HR=0.73, p=0.038). GGR levels were reflected in an unsupervised clustering of metabolomics profiles. In a supervised analysis of metabolomics data and of genome-wide expression data, replacement of GGR by metabolite surrogate markers was feasible, while replacement of GGR by RNA markers had a limited accuracy. Functional analysis of the gene expression data showed negative correlation between glutamate enrichment and activation of peroxisome proliferator-activated receptor (PPAR) pathway. Our findings may have important implications for patient stratification related to utilization of glutaminase inhibitors. © 2014 Wiley Periodicals, Inc.