Objective: Gastroesophageal reflux disease (GERD) clinically predisposes to columnar Barrett's metaplasia (BM) in the distal esophagus. We demonstrate evidence supporting cellular origin of BM from reprograming or trans-commitment of resident normal esophageal squamous (NES) epithelial cells in response to acid and bile exposure using an in vitro cell culture model. Design: The hTERT-immortalized NES cell line, NES-B10T, was exposed 5 min/day to Acid and Bile (A+B) mixture for 30 weeks. Morphological changes, mRNA and protein expression levels for inflammatory marker, COX-2; lineage determining transcription factors, TAp63 (Squamous), CDX2 and SOX9 (both columnar); and markers of columnar lineage, Villin, Muc-2, CK8, and mAb Das-1 (incomplete phenotype of intestinal metaplasia) were assessed every 10 weeks. Results: Markers of columnar lineage, and inflammation increased progressively, while squamous lineage determining transcriptional factors were significantly decreased both at the mRNA and/or protein level in the NES-B10T cells at/after acid and bile treatment for 30 weeks. Distinct modifications in morphological features were only observed at/after 30 weeks of A+B exposure. These changes acquired by the NES-B10T 30 week cells were retained even after cessation of A+B exposure for at least 3 weeks. Conclusions: This study provides evidence that chronic exposure to physiological components of gastric refluxate led to repression of the discernable squamous transcriptional factors and activation of latent columnar transcriptional factors. This reflects the alteration in lineage commitment of the precursor-like bi-phenotypic, NES-B10T cells in response to A+B exposure as the possible origin of BM from the resident NES cells.