Metformin is under evaluation as a potential anticancer agent. Expression of total and phospho(Thr172)-adenosine monophosphate-activated kinase-α (AMPKα and pAMPKα(Thr172) respectively), a main metformin target, was examined in radiotherapy treated breast cancers and metformin's ability to modulate Trx system expression and breast cancer radiosensitivity evaluated in vitro. AMPKα and pAMPKα(Thr172) expression was assessed using a discovery (n=166) and validation cohort (n=609). Metformin's role in regulating radioresponse, and Trx family expression, was examined via clonogenic assays and Western blots. Intracellular reactive oxygen species (ROS) levels, cell cycle progression and apoptosis were assessed by flow cytometry. High AMPKα expression associated with improved local recurrence-free (P=0.019), relapse-free (P=0.016) and breast cancer-specific survival (P=0.000065) and was, from multivariate analysis, an independent prognostic factor from the discovery cohort. From the validation cases AMPKα expression associated with relapse-free and breast cancer-specific survival in luminal breast cancers. Metformin substantially increased radiosensitivity, intracellular ROS levels and reduced Trx expression, in luminal breast cancer cells, but had little effect on basal phenotype cells. In conclusion, high AMPKα expression associates with improved prognosis, especially in luminal breast cancer. Metformin preferentially radiosensitises luminal breast cancer cells, potentially due to alterations to intracellular ROS levels via modulation of Trx family protein expression.