Repurposing metformin for cancer therapy is attractive due to its safety profile, epidemiological evidence, and encouraging data from human clinical trials. Although it is known to systemically affect glucose metabolism in liver, muscle, gut, and other tissues, the molecular determinants that predict a patient response in cancer remain unknown. Here, we carry out an integrative metabolomics analysis of metformin action in ovarian cancer. Metformin accumulated in patient biopsies, and pathways involving nucleotide metabolism, redox, and energy status, all related to mitochondrial metabolism, were affected in treated tumors. Strikingly, a metabolic signature obtained from a patient with an exceptional clinical outcome mirrored that of a responsive animal tumor. Mechanistically, we demonstrate with stable isotope tracing that these metabolic signatures are due to an inability to adapt nutrient utilization in the mitochondria. This analysis provides new insights into mitochondrial metabolism and may lead to more precise indications of metformin in cancer. Display omitted •Metformin accumulates in human tumor biopsies at micromolar concentrations•The response to metformin in humans can be modeled in nutrient-limited environments•Mitochondrial substrate utilization underlies metformin sensitivity•Restoration of specific mitochondrial outputs causes resistance to metformin Liu et al. carry out an integrative metabolomics analysis of metformin action in ovarian cancer. Using patient samples (including those from a “super-responder”), animal tumors, and cell culture models, they find that the predominant mechanism of action by metformin in cancer is to target tumor-cell-intrinsic mitochondrial metabolism.