The pathogenesis of reflux esophagitis is not well understood and remains controversial. Distal gastrectomy serves as a model to assess the role of duodenal reflux with low gastric acidity in the development of reflux esophagitis. We investigated the relationship between the severity of esophagitis and gastroduodenal juice reflux, with particular focus on trypsin and bile acids after distal gastrectomy reconstructed with Billroth I anastomosis. Twenty-eight patients with gastroesophageal reflux disease after distal gastrectomy were enrolled. Esophageal and duodenal contents were aspirated under endoscopical examination, and their trypsin activity and bile acid concentrations were measured. The grade of reflux esophagitis was assessed by endoscopy and the symptoms were scored. Moreover, the grade of infiltration of inflammatory cells and the expression of COX-2 mRNA in the esophageal epithelium were evaluated. Patients with severe esophagitis had a higher amount of trypsin activity and bile acid concentrations in the esophagus, but not in the duodenum, compared to patients with mild esophagitis (P < 0.05). There was a strong positive correlation between the trypsin activity and the bile acid concentrations in the esophagus (r = 0.743, P = 0.0001). Moreover, the COX-2 mRNA expression and the grade of infiltrating inflammatory cells in the esophageal mucosa significantly correlated with the trypsin activity and bile acid concentrations in the esophagus. Thus, duodenogastroesophageal reflux with low gastric acidity is one of the pathogeneses in the development of reflux esophagitis from the present clinical study with patients after distal gastrectomy reconstructed with Billroth I anastomosis.