Estrogen plays an important role in breast cancer development. While the mechanism of the estrogen effects is not fully elucidated, one possible route is by increasing the stem cell-like properties in the tumors. Tocopherols are known to reduce breast cancer development and progression. The aim of the present study is to investigate the effects of tocopherols on the regulation of breast cancer stemness mediated by estrogen. To determine the effects of tocopherols on estrogen-influenced breast cancer stem cells, the MCF-7 tumorsphere culture system, which enriches for mammary progenitor cells and putative breast cancer stem cells, was utilized. Treatment with estrogen resulted in an increase in the CD44+/CD24- subpopulation and aldehyde dehydrogenase activity in tumorspheres as well as the number and size of tumorspheres. Tocopherols inhibited the estrogen-induced expansion of the breast cancer stem population. Tocopherols decreased the levels of stem cell markers, including octamer-binding transcription factor 4 (OCT4), CD44 and SOX-2, as well as estrogen-related markers, such as trefoil factor (TFF)/pS2, cathepsin D, progesterone receptor and SERPINA1, in estrogen-stimulated tumorspheres. Overexpression of OCT4 increased CD44 and sex-determining region Y-box-2 levels and significantly increased cell invasion and expression of the invasion markers, matrix metalloproteinases, tissue inhibitors of metalloproteinase and urokinase plasminogen activator, and tocopherols inhibited these OCT4-mediated effects. These results suggest a potential inhibitory mechanism of tocopherols in estrogen-induced stemness and cell invasion in breast cancer.