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Esophageal Adenocarcinoma is the fastest growing cancer in the United States and is the sixth leading cause of cancer-related death. In its precancerous lesion, Barrett’s Esophagus, the normal squamous epithelium of the esophagus undergoes columnar metaplasia due to long-term exposure to reflux contents of which bile acid at acidic pH is the chief component. Presently, there is no simple in vitro culture system of human esophageal cells that can be used to observe the morphological and molecular effects of bile acid and low pH on a stratified epithelium. In this study, we showed that h-TERT-transformed primary esophageal squamous cells (EPC1) form a 10-11 layered stratified epithelium when grown on polyester trans-well filters apically and basally supplemented with keratinocyte serum-free media with 0.6mM Ca+2. This stratified epithelium shows epithelial barrier function and expresses squamous specific genes like GRHL-1, K10, KDAP, DSG1, and IVL. Moreover, when exposed to bile acids at pH5 in short pulses, EPC1 cells demonstrate reduction in the stratification layers and in the expression of squamous specific genes. The epithelium also exhibits loss of barrier function possibly due to disruption of desmosomal junctions and phosphorylation-activation of epidermal growth factor receptor (EGFR) and down-stream pathways. In addition, the epithelium starts expressing columnar specific transcription factor CDX2 as early as day 3 of treatment. These results indicate that bile acid at low pH is responsible for skewing the differentiation status of stratified squamous esophageal epithelium in vitro to a more columnar type possibly by initiating a mucosal restitution response through activation of EGFR signaling.