The aim of the present study was to assess the impact of a Rhei Rhizoma and Citri Pericarpium (RC) mixture on esophageal mucosal injury in rats with acute reflux esophagitis (ARE). An experimental model of ARE was established surgically. RC mixture was administered at a dose of 100 or 200 mg/kg body weight 90 min prior to induction of ARE, and its effects were compared with those of the Control group, which underwent the ARE procedure without treatment. Administration of the RC mixture significantly ameliorated the severity of the histopathological changes compared with the ARE control group. Elevated levels of reactive oxygen species (ROS) in the esophageal tissues of reflux esophagitis-induced rats was significantly decreased by the RC mixture. Moreover, the increased ROS production induced by the increase in NADPH oxidase 4 (NOX4) and p22phox was down-regulated by the RC mixture. The protein expression of inflammatory mediators and cytokines together with nuclear factor kappa B (NF-κB) through the degradation of inhibitor of NF-κB was modulated by the RC mixture treatment. The levels of the anti-inflammatory cytokines, such as interleukin (IL)-4 and IL-10, were significantly increased. Additionally, RC mixture supplementation improved esophageal barrier function through upregulating claudin −1, −3 and −4 protein expression levels. Taken together, these results suggest that RC mixture treatment may attenuate the esophageal mucosal injury, possibly through suppression of ROS formation, inhibiting NF-κB inflammatory signaling, and up-regulating the expression of proteins associated with tight junction formation, including claudin −1, −3 and −4.