Esophageal adenocarcinoma (EA) has increased in incidence over the past several decades and is characterized by a poor prognosis. Gastroesophageal reflux disease (GERD) complicated by Barrett’s esophagus (BE) is a major risk factor for the development of EA. There is a progression from BE to dysplasia and to EA. However, the mechanisms of progression from BE to EA are not fully understood. We found that NOX5-S is present in Barrett’s cells BAR-T and EA cells FLO and OE33 and is overexpressed in FLO cells and EA tissues. NOX5-S mRNA is also increased in Barrett’s mucosa with high-grade dysplasia. Pulsed acid treatment significantly increases NOX5-S expression and H2O2 production in BAR-T and OE33 cells and Barrett’s mucosa. These data suggest that NOX5-S may be a source of overproduction of reactive oxygen species (ROS) in BE and in EA cells. We also found that acid-induced increase in NOX5-S expression may depend on activation of Rho kinase, ERK1/2 MAP kinases, and cAMP response element-binding protein. An acid may also induce production of platelet-activating factor, which activates signal transducer and activator of transcription 5 (STAT5) and then upregulates NOX5-S. In addition, NOX5-S mediates acid-induced increase in cell proliferation in Barrett’s cells BAR-T and EA cells (OE33 and FLO). NOX5-S-mediated increase in cell proliferation may depend on activation of COX2 and microsomal prostaglandin E synthase 1 (mPGES1), and downregulation of p16 via promoter methylation. NOX5-S also mediates acid-induced DNA damage. These data suggest that persistent acid reflux present in BE patients may upregulate NOX5-S, increase production of ROS and cell proliferation, and cause DNA damage, thereby contributing to the progression from BE to dysplasia and to EA.