A novel melatonin, estrogen, and progesterone hormone therapy was developed as a safe bio-identical alternative hormone therapy for menopausal women based on the Women's Health Initiative findings that PremPro™ increased breast cancer risk and mortality of all types of breast cancer in postmenopausal women. For HER2 breast cancer, melatonin, estrogen, and progesterone delayed tumor onset and reduced tumor incidence in female mice. For other breast cancers, its actions are unknown. In this study, melatonin, estrogen, and progesterone hormone therapy were assessed in human ER+ (MCF-7) and triple negative breast cancer (MDA-MB-231) cells, and found to decrease proliferation and migration of both breast cancer lines. Inhibition of MEK1/2 and 5 using PD98059 and BIX02189, respectively, inhibited proliferation and migration in MDA-MB-231 cells and proliferation in MCF-7 cells; however, when combined with melatonin, estrogen, and progesterone, BIX02189 blocked melatonin, estrogen, and progesterone-mediated inhibition of migration in MCF-7 cells and induced Elf-5. For MDA-MB-231 cells, BIX02189 combined with melatonin, estrogen, and progesterone inhibited proliferation and increased pERK1/2 and β1-INTEGRIN; levels of pERK5 remained low/nearly absent in both breast cancer lines. These findings demonstrate novel anti-cancer actions of melatonin, estrogen, and progesterone in ER+ and triple negative breast cancer cells through intricate MEK1/2- and MEK5-associated signaling cascades that favor anti-proliferation and anti-migration.