Metformin, one of the most widely prescribed anti-hyperglycemic drugs, has recently received increasing attention for its potential effects with regard to cancer prevention and treatment. However, the mechanisms behind the suppression of cancer cell growth by metformin remain far from completely understood. The aim of the present study was to investigate whether metformin could regulate histone modification and its downstream gene transcription, and its potential function in inhibiting breast cancer cell proliferation. A T47D cell proliferation curve was determined by cell counting following metformin treatment with differing doses or time courses. The cell cycle was analyzed by flow cytometry with propidium iodide staining. Histone H2B monoubiquitination was evaluated by western blotting subsequent to histone extraction. The histone H2B monoubiquitination downstream gene expression level was determined by quantitative PCR. The results showed that metformin changed the cell-cycle check-point and inhibited breast cancer cell proliferation in a dose-dependent manner. AMPK was activated and histone H2B monoubiquitination and downstream gene transcription were inhibited following metformin treatment in the T47D cells. The effect of metformin on T47D cell proliferation was dependent on AMPK activity. It was concluded that metformin can suppress breast cancer cell growth by the activation of AMPK and the inhibition of histone H2B monoubiquitination and downstream gene transcription. This study reveals a novel potential mechanism of cancer cell growth suppression by metformin.