In this study, we have established a large-scale profile of promoter methylation in Chinese ESCC patients. We combined analysis the Infinium HD 450K methylation array data with gene expression array data. From this we identified a set of genes that have potential functional consequences owing to the aberrant promoter DNA methylation. The pathway analysis showed that these genes take part in tumor-related pathways which may play an important role in the development and progression of the disease. We chose 3 genes from 168 genes for validation in independent tissue samples and evaluated their significance in clinical diagnosis of ESCC. Therefore, our results not only strengthen the findings on the importanance of aberrant DNA methylation in ESCC, but also provide a novel and more comprehensive signature of ESCC methylation. We tested DNA methylation in more than 480,000 CpG sites using the Infinium HD 450K methylation array in ESCC samples, adjacent normal LY2157299 surrounding tissues, and normal mucosa of healthy individuals. An initial, unsupervised hierarchical analysis with all of the tested CpG sites showed that there are two main clusters: tumor and normal . This indicates that tumors show different methylation profiles in comparison with normal tissues. Interestingly, our result also shows that adjacent normal surrounding tissues also have different DNA methylome patterns compared with normal tissues. We presume there may be field cancerization phenomenon existed in adjacent normal tissues. More studies need to be done to explore the detail mechanism of methylation difference between normal and adjacent normal tissues. BRB-Array Tools is an integrated software system for the comprehensive analysis of DNA microarray experiments. By class comparison using the BRB-Array Tools, we identified 66,857 differentially methylated CpG sites in ESCC. We chose to limit our analysis to the proximal promoter region because this region is well characterized for its effects of DNA methylation on gene silencing. This approach also allowed us to evaluate associations with gene expression changes based on an independent ESCC data set. The assumption is that inverse correlations between promoter methylation and gene expression may plausibly indicate a functional result of differential methylated genes identified in ESCC. This approach identified 168 genes in ESCC. Based on the combined analysis signature of methylation data and expression data, we identified the pathways that are specifically altered in this type of cancer. Our results suggested that these altered pathways may play pivotal role in ESCC tumorigenesis. Restoration of EPB41L3 expression in non-small cell lung cancer or breast cancer cell lines significantly suppressed cell growth in vitro, and re-expression of EPB41L3 can induce extensive apoptotic cell death in ovarian cancer cells. Recent studies show that the promoter methylation of EPB41L3, leading to loss of its expression, is an important molecular event in several types of tumor cells, whereas EPB41L3 expression can be restored by a demethylating agent.