The mechanism by which gastroesophageal reflux promotes metaplasia→dysplasia→carcinoma is unknown. The aim of the study is to determine if repeated exposure to acid and bile confers a tumorigenic phenotype in a telomerase (hTERT)-immortalized benign Barrett's cell line (BAR-T). BAR-T cells were exposed to acid (pH 4) (A) and bile salt (200 μM glycochenodeoxycholic acid) (B) daily for 5 min up to 65+ weeks. The control cells were grown in parallel without any A or B treatment. Cell morphology, proliferation, transformation, and molecular changes in the gene expression for COX-2, TC22, p53 and p53 target genes were analyzed at 8-12 weeks intervals. At 46 weeks BAR-T cells exposed to (A+B) showed distinct phenotypic changes: forming clusters and acini, and at 65 weeks displayed foci in monolayer, and formed distinct colonies in soft agar. Untreated cells did not show any such changes. In A+B-treated BAR-T cells, COX-2 mRNA increased 10- to 20-fold, TC22 mRNA increased by 2- to 3-fold at 22-65 weeks, p53, MDM2, PERP, and p21mRNA increased 2.5-, 6.4-, 4-, and 2.6-fold respectively when compared to untreated cells at 34 weeks. However, at 58 weeks onward, there was a sharp decline of p53 and its target genes to the baseline level. At 65 weeks A+B-treated BAR-T cells formed tumor in nude mice whereas untreated cells did not. We demonstrate a novel in vitro model of transformation of a benign Barrett's cell line following repeated exposure to A+B over the course of 65 weeks.