Hydrophobic bile acids like deoxycholic acid (DCA), which cause oxidative DNA damage and activate NF-B in Barrett's metaplasia, might contribute to carcinogenesis in Barrett's esophagus. We have explored mechanisms whereby ursodeoxycholic acid (UDCA, a hydrophilic bile acid) protects against DCA-induced injury in vivo in patients and in vitro using non-neoplastic, telomerase-immortalized Barrett's cell lines. We took biopsies of Barrett's esophagus from 21 patients before and after esophageal perfusion with DCA (250 µM) at baseline, and after 8 weeks of oral UDCA treatment. DNA damage was assessed by phospho-H2AX expression, neutral comet assay and phospho-H2AX nuclear foci formation. Quantitative PCR was performed for antioxidants including catalase and GPX1. Nrf2, catalase, and GPX1 were knocked down with siRNAs. Reporter assays were performed using a plasmid construct containing antioxidant responsive element. In patients, baseline esophageal perfusion with DCA significantly increased phospho-H2AX and phospho-p65 in Barrett's metaplasia. Oral UDCA increased GPX1 and catalase levels in Barrett's metaplasia, and prevented DCA perfusion from inducing DNA damage and NF-B activation. In cells, DCA-induced DNA damage and NF-B activation was prevented by 24-hour pretreatment with UDCA, but not by mixing UDCA with DCA. UDCA activated Nrf2 signaling to increase GPX1 and catalase expression, and protective effects of UDCA pretreatment were blocked by siRNA knockdown of these antioxidants. UDCA increases expression of antioxidants that prevent toxic bile acids from causing DNA damage and NF-B activation in Barrett's metaplasia. Elucidation of this molecular pathway for UDCA protection provides rationale for clinical trials on UDCA for chemoprevention in Barrett's esophagus.