Objectives <p>This study aims to explore the functions and mechanisms underlying the involvement of 14-3-3β in esophageal squamous cell carcinoma (ESCC).</p> Methods <p>Western blot and quantitative real-time PCR (RT-PCR) were utilized to evaluate the levels of 14-3-3β in SHEE, TE-1, and KYSE-150 cells. Following infection with YWHAB-RNAi lentivirus and puromycin screening, the reduction of 14-3-3β expression in KYSE-150 cells was confirmed via western blot and RT-PCR. Subsequently, wound healing, CCK8, and colony-formation assays were performed to assess cell migration and proliferation in KYSE-150 cells with stable low levels of 14-3-3β. Furthermore, western blot analysis was conducted to examine the expression of relevant proteins, elucidating the potential molecular mechanisms underlying 14-3-3β involvement in ESCC progression and metastasis.</p> Results <p>The expression of 14-3-3β was significantly elevated in KYSE-150 cells compared to SHEE cells. Upon infection in KYSE-150 cells, the expression of 14-3-3β was diminished, resulting in suppressed cell migration and colony formation, although no significant changes were observed in the CCK8 assays. Furthermore, in KYSE-150 cells exhibiting reduced 14-3-3β expression, the activity of phosphorylated AKT (p-AKT) was significantly decreased, while no notable difference was detected in AKT protein levels. Additionally, there were no significant alterations observed in apoptosis-related proteins, including BCL2, BAX, Caspase3, and activated Caspase9.</p> Conclusions <p>In KYSE-150 cells infected with YWHAB-RNAi lentivirus, the downregulation of 14-3-3β expression resulted in significant reductions in both cell migration and proliferation. Furthermore, the findings demonstrated that 14-3-3β may regulate the biological activities of ESCC cells via modulation of the PI3K/AKT signaling pathway.</p>