Natural components continue to be an important source for the discovery and development of novel anticancer agents. Polymethoxyflavones are a class of flavonoids found in citrus fruits and medicinal plants used in traditional medicine. In the present study, the anticancer activity of the well-known nobiletin (5,6,7,8,3',4'-hexamethoxyflavone) was compared against its less studied structural isomer 5,6,7,3',4',5'-hexamethoxyflavone. These compounds were evaluated on the Hs578T triple-negative breast cancer cell line and its more migratory subclone Hs578Ts(i)8. 5,6,7,3',4',5'-hexamethoxyflavone was found to be less toxic than nobiletin, while a similar growth inhibitory effect was observed after 72 h. Additionally, 5,6,7,3',4',5'-hexamethoxyflavone arrested the cell cycle in the G2/M phase, while no effect was observed on apoptosis or the migratory behavior of these cells. Furthermore, mechanistic studies revealed that the growth inhibition was concomitant with reduced phosphorylation levels of signaling molecules in the MAPK and Akt pathways as well as cell cycle regulators, involved in regulating cell proliferation, survival and cell cycle. In summary, the present study is the first to report on the anticancer activities of 5,6,7,3',4',5'-hexamethoxyflavone and to provide evidence that this flavone could have a greater potential than nobiletin for prevention or treatment of triple- negative breast cancer.