Metabolic reprogramming has been proposed to be a hallmark of cancer, yet a systematic characterization of the metabolic pathways active in transformed cells is currently lacking. Using mass spectrometry, we measured the consumption and release (CORE) profiles of 219 metabolites from media across the NCI-60 cancer cell lines, and integrated these data with a preexisting atlas of gene expression. This analysis identified glycine consumption and expression of the mitochondrial glycine biosynthetic pathway as strongly correlated with rates of proliferation across cancer cells. Antagonizing glycine uptake and its mitochondrial biosynthesis preferentially impaired rapidly proliferating cells. Moreover, higher expression of this pathway was associated with greater mortality in breast cancer patients. Increased reliance on glycine may represent a metabolic vulnerability for selectively targeting rapid cancer cell proliferation.