This study was designed to determine the significance of DNA methyltransferases 
(DNMTs) in DNA hypermethylation in esophageal squamous cell carcinoma (ESCC) and 
to identify DNA methylation markers in serum for the early diagnosis of ESCC. A 
promoter methylation profile of 12 tumor-related genes was assessed using 
methylation-specific PCR in ESCC and paired non-tumor tissue samples from 47 
patients. Expression levels of DNMTs were examined by real-time reverse 
transcription-PCR and immunohistochemistry. Using MethyLight, the methylation 
status of 5 genes was analyzed in serum samples from 45 patients and 15 healthy 
individuals. A total of 46 (97.9%) of 47 ESCC samples showed methylation in at 
least one of the examined genes, and methylation was most frequent for RAR-β 
(46.8%), DAPK (46.8%), p16 (44.7%), and CDH1 (42.6%). Methylation of RASSF1A was 
significantly correlated with the poorly differentiated tumors and the early 
pathologic tumor classification (P=0.035 and P=0.046, respectively). Tumoral 
DNMT3b mRNA up-regulation was significantly correlated with hypermethylation of 
multiple tumor-related genes (P=0.021). In addition, hypermethylation of 
cell-free serum DNA was common in ESCC patients, and diagnostic accuracy was 
increased when methylation of multiple genes (RAR-β, DAPK, CDH1, p16 and 
RASSF1A) were analyzed in combination (ROC AUC 0.911, 82.2% sensitivity and 100% 
specificity). The present study suggests that hypermethylation of multiple 
tumor-related genes may be involved in the pathogenesis of ESCC and mediated by 
the increase of DNMT3b expression. A cluster of multiple methylated genes in 
serum DNA has the potential as a novel biomarker for ESCC diagnosis.