Gastroesophageal reflux disease (GERD) is associated with impaired epithelial barrier function. However, the influence of acid and/or bile acids on human esophageal epithelial barrier function and the tight junction (TJ) proteins has not been fully elucidated. The aim of the study is to investigate the esophageal barrier function and TJ expression in healthy subjects and patients with GERD. The functionality of esophageal mucosa exposed to bile salt deoxycholic acid (DCA) and trypsin has been studied in vitro. Endoscopic biopsies from healthy controls and patients with GERD-related symptom with endoscopic erosive signs, as well as esophageal mucosa taken from patients undergoing esophagectomy were evaluated in Ussing chambers and by western blot and immunohistochemistry. The esophageal epithelium from GERD patients had lower electrical resistance and higher epithelial currents than controls. Claudin-1 and -4 were significantly decreased in GERD patients. The bile salt DCA in the low concentration of 1.5 mM and trypsin increased the resistance and claudin-1 expression, while the higher concentration of 2.5 mM DCA and trypsin decreased the resistance and the claudin-3, -4 and E-cadherin expressions. In addition to acidic reflux, duodenal reflux components, such as bile salts and trypsin, have the potential to disrupt the esophageal barrier function, partly by modulating the TJ proteins. However, the expression of TJ is dependent on both the refluxed material as well as the concentration of the bile salt.