Tumor interstitial fluid (TIF) contains the tumor secretome and forms a critical component of the tumor microenvironment. Cyclooxygenase-2 (COX2) mediates the inflammatory response of cells and is upregulated in cancers. In cancers, COX-2 expression has been related to increased invasion and metastasis. Here, for the first time, using 1H MR spectroscopy we characterized changes in the metabolic patterns of TIF in tumors derived from triple negative SUM-149 human breast cancer cells with COX-2 overexpressed. COX-2 overexpression significantly altered several fundamental metabolic pathways. These data provide new insights into the role of COX-2 in tumor aggressiveness, and identify new metabolic targets.