Objective: Esophageal cancer (EC) is an aggressive malignancy associated with a high mortality rate, and metastasis is one of the main causes of death in patients with EC. High cholesterol is positively associated with the risk of developing EC. 27-Hydroxycholesterol (27-HC) is the most abundant cholesterol metabolite. Here, we investigated the role of cholesterol and 27-HC in EC cell migration. Methods: The effects of cholesterol and 27-HC on EC cell migration were examined using migration assays in the human esophageal squamous cell line ECA109 and the human esophageal adenocarcinoma cell line OE33. Transient transfection and western blot analysis were performed to examine the mechanisms underlying the modulation of EC cell migration by glycogen synthase kinase-3-beta (GSK‑3β)/β‑catenin signaling. Results: Cholesterol induced epithelial-to-mesenchymal transition (EMT) and promoted the migration of EC cells. The enzyme CYP27A1, which catalyzes the conversion of cholesterol to 27-HC, promoted EC cell EMT and migration. 27-HC increased the production of reactive oxygen species (ROS), which promoted the phosphorylation and activation of the AKT and p38 signaling pathways. Phosphorylated AKT inhibited GSK-3β/β-catenin binding, which increased the free β-catenin level and its nuclear translocation. This process together with the activation of p-p38-related pathways promoted EMT and increased the migratory ability of EC cells. Conclusions: ROS production induced by cholesterol and its metabolite 27-HC activated the AKT signaling pathway and promoted the translocation of β-catenin into the nucleus, which induced EMT and increased the migration of EC cells. The p38 kinase induced the migration of EC cells through a mechanism other than β-catenin signaling.