Population studies have revealed that treatment with the anti-diabetic drug metformin significantly associates with reduced breast cancer risk.  Animal studies have shown that metformin suppresses the development of mammary carcinomas in transgenic female mice carrying a HER2 oncogene, but not that of spontaneous tumors.  We herein demonstrate that HER2 oncoprotein itself may represent a key cellular target involved in the anti-breast cancer actions of metformin.  First, ectopical overexpression of HER2 oncogene significantly enhances metformin-induced breast cancer cell growth inhibition.  Second, metformin treatment drastically down-regulates HER2 protein levels (up to 85% reduction) in a dose- and time-dependent manner. Metformin-induced inhibition of HER2 take places regardless the molecular mechanism contributing to HER2 overexpression (i.e., human HER2 cDNA exogenously driven by a viral promoter and naturally occurring endogenous HER2 gene amplification).  Mechanistically, metformin-induced suppression of HER2 overexpression appears to occur via direct (AMPK-independent) inhibition of p70S6K1 activity.  Compound C- and small interference RNA (siRNA)-induced blockade of AMPK activity/expression fail to prevent the anti-HER2 effect of metformin while AMPK hyperactivation following exposure to the AMP analog AICAR is not sufficient to down-regulate HER2 expression. HER2-positive breast cancer cells transfected with p70S6K1 siRNA become completely refractory to metformin-induced HER2 suppression. Of note, co-incubation with agents that block reactive oxygen species (ROS) production (e.g. N-acetylcisteine) dramatically enhanced the ability of metformin to decrease HER2 expression. From the perspective of chemoprevention, these findings altogether suggest that metformin might exert a protective mostly confined to the HER2-positive breast cancer subtype. From the perspective of intervention, the presence/absence of molecular hallmarks such as HER2 overexpression and/or p70S6K1 hyperactivation might dictate alternative responses in metformin-based treatment of early breast cancer. The importance of mTOR/p70S6K1-sensed ROS status at mediating the anti-oncogenic effects of metformin might represent a previously unrecognized linkage molecularly connecting its anti-aging and anti-cancer actions.