Chronic gastroesophageal reflux disease is a known risk factor for Barrett's esophagus (BE), which induces oxidative mucosal damage. Glutathione peroxidase-3 (GPx3) is a secretory protein with potent extracellular antioxidant activity. In this study, we have investigated the mRNA and protein expression of GPx3, and explored promoter hypermethylation as an epigenetic mechanism for GPx3 gene inactivation during Barrett's carcinogenesis. Quantitative real-time reverse transcription polymerase chain reaction on 42 Barrett's adenocarcinomas (BAs) revealed consistently reduced levels of GPx3 mRNA in 91% of tumor samples. GPx3 promoter hypermethylation was detected in 62% of Barrett's metaplasia, 82% of dysplasia, and 88% of BA samples. Hypermethylation of both alleles of GPx3 was most frequently seen in BAs (P = .001). Immunohistochemical staining of GPx3 in matching tissue sections (normal, BE, Barrett's dysplasia, and BA) revealed strong immunostaining for GPx3 in normal esophageal and gastric tissues. However, weak to absent GPx3 staining was observed in Barrett's dysplasia and adenocarcinoma samples where the promoter was hypermethylated. The degree of loss of immunohistochemistry correlated with the hypermethylation pattern (monoallelic versus biallelic). The observed high frequency of promoter hypermethylation and progressive loss of GPx3 expression in BA and its associated lesions, together with its known function as a potent antioxidant, suggest that epigenetic inactivation and regulation of glutathione pathway may be critical in the development and progression of BE.