Late-phase clinical trials investigating metformin as a cancer therapy are underway. However, there remains controversy as to the mode of action of metformin in tumors at clinical doses. We conducted a clinical study integrating measurement of markers of systemic metabolism, dynamic FDG-PET-CT, transcriptomics, and metabolomics at paired time points to profile the bioactivity of metformin in primary breast cancer. We show metformin reduces the levels of mitochondrial metabolites, activates multiple mitochondrial metabolic pathways, and increases 18-FDG flux in tumors. Two tumor groups are identified with distinct metabolic responses, an OXPHOS transcriptional response (OTR) group for which there is an increase in OXPHOS gene transcription and an FDG response group with increased 18-FDG uptake. Increase in proliferation, as measured by a validated proliferation signature, suggested that patients in the OTR group were resistant to metformin treatment. We conclude that mitochondrial response to metformin in primary breast cancer may define anti-tumor effect.