Esophageal squamous cell carcinoma (ESCC) is one of the most common causes of cancer mortality in the gastrointestinal tract. Promoter hypermethylation of tumor suppressor genes contributes to gene inactivation during development of ESCC. To identify novel methylation-silenced genes in ESCC. Genome-wide microarrays were applied to search for genes that were markedly upregulated after treatment with 5-aza-2'-deoxycytidine (5-Aza-dC) and that were markedly decreased in tumor tissue compared with paired adjacent nontumor tissue. Reverse-transcription polymerase chain reaction (PCR), immunohistochemistry, methylation-specific PCR, and bisulfite genomic sequencing were employed to investigate expression and methylation of candidate genes in five human ESCC cell lines, two human immortalized normal esophageal epithelial cell lines, primary ESCC tumor tissues, and paired adjacent nontumor tissues. GPX3 was selected as a novel candidate hypermethylated gene in ESCC through microarray analysis. In most ESCC cell lines, GPX3 messenger RNA (mRNA) expression was downregulated and the CpG island of GPX3 promoter was methylated. Demethylation treatment with 5-Aza-dC restored GPX3 mRNA expression. Methylation of GPX3 promoter was more frequent in ESCC tumor tissues (71.4%) than in adjacent nontumor tissues (10.7%) (P < 0.001), and methylation of GPX3 promoter correlated significantly with GPX3 mRNA downregulation. Finally, GPX3 protein expression was also significantly lower in ESCC tumor tissues than in adjacent nontumor tissues. GPX3 is downregulated through promoter hypermethylation in ESCC, which may be a potential biomarker of ESCC.