SLC26A3 encodes a Cl-/HCO3(-) ion transporter that is also known as downregulated in adenoma (DRA) and is involved in HCO3-/mucus formation. The role of DRA in the epithelial barrier has not been previously established. In this study, we investigated the in vivo and in vitro mechanisms of DRA in the colon epithelial barrier. Immunofluorescence (IF) and coimmunoprecipitation (co-IP) studies reveal that DRA binds directly to tight junction (TJ) proteins and affects the expression of TJ proteins in polarized Caco-2BBe cells. Similarly, DRA colocalizes with ZO-1 in the intestinal epithelium. Knockdown or overexpression of DRA leads to alterations in TJ proteins and epithelial permeability. In addition, TNF-alpha treatment downregulates DRA by activating NF-kappa B and subsequently affecting intestinal epithelial barrier integrity. Furthermore, overexpression of DRA partly reverses the TNF-alpha-induced damage by stabilizing TJ proteins. Neutralization of TNF-alpha in dextran sulfate sodium (DSS)-induced colitis mice demonstrates improved the outcomes, and the therapeutic effect of the TNF-alpha neutralizing mAb is mediated in part by the preservation of DRA expression. These data suggest that DRA may be one of the therapeutic targets of TNF-alpha. Moreover, DRA delivered by adenovirus vector significantly prevents the exacerbation of colitis and improves epithelial barrier function by promoting the recovery of TJ proteins in DSS-treated mice. In conclusion, DRA plays a role in protecting the epithelial barrier and may be a therapeutic target in gut homeostasis.