Most tumors exhibit increased glucose metabolism to lactate, however, the extent to which glucose-derived metabolic fluxes are used for alternative processes is poorly understood. Using a metabolomics approach with isotope labeling, we found that in some cancer cells a relatively large amount of glycolytic carbon is diverted into serine and glycine metabolism through phosphoglycerate dehydrogenase (PHGDH). An analysis of human cancers showed that PHGDH is recurrently amplified in a genomic region of focal copy number gain most commonly found in melanoma. Decreasing PHGDH expression impaired proliferation in amplified cell lines. Increased expression was also associated with breast cancer subtypes, and ectopic expression of PHGDH in mammary epithelial cells disrupted acinar morphogenesis and induced other phenotypic alterations that may predispose cells to transformation. Our findings show that the diversion of glycolytic flux into a specific alternate pathway can be selected during tumor development and may contribute to the pathogenesis of human cancer.