Triple-positive breast cancer (TPBC) is a type of breast cancer that overexpresses estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). Dysregulation of ER signaling has been implicated in the pathogenesis of breast cancer. ERα activation triggers the production of second messengers, including cAMP, leading to the activation of signals such as PI3K/AKT or Ras/MAPK. Ruxolitinib is a specific inhibitor of JAK1/JAK2. MK-2206 is an allosteric inhibitor of the Akt. The limitations of the use of ruxolitinib and MK-2206 as single agents necessitate the development of combination therapies with other drugs. This study is the first to investigate the effects of combining ruxolitinib with MK-2206 on MAPK and PI3K/AKT signaling in BT474 breast cancer cells. In addition, this work aimed to increase the anticancer effects of cotreatment with MK-2206 and ruxolitinib. Ruxolitinib, MK-2206, and their combination reduced cell viability in a dose- and time-dependent manner, as determined by MTT assays after 48 h of treatment. Colony formation and wound healing assays demonstrated that MK-2206 exhibited a synergistic anti-proliferative effect. The effects of ruxolitinib, MK-2206, and their combination on PI3K/AKT and MAPK signaling were assessed via western blotting. Ruxolitinib and MK-2206 combined treatment inhibit cell death in BT474 cells by downregulating ERα, Src-1, ERK1/2, SAPK/JNK, and c-Jun. Our results revealed the relationships among the ERα, PI3K/AKT, and MAPK signaling pathways in ER+ breast cancer cells. Understanding the interactions among ERα, PI3K-AKT-mTOR, and MAPK could lead to novel combination therapies.