Esophageal squamous cell carcinoma (ESCC) is one of the most common tumors in human. Previous studies showed that multiple genetic and epigenetic alterations involved in carcinogenesis of esophagus, whereas the molecular mechanisms are poorly understood. So far, more and more ESCC-related genes have been found and retinoic acid receptor β₂ (RARβ₂) is such a gene which was recognized as a putative tumor suppressor gene since reduced RARβ₂ mRNA expression has been observed in several solid tumors, including ESCC. A growing evidence indicated that RARβ₂ was required for the growth inhibitory effect of retinoic acid (RA). However, the molecular mechanism of its inactivation remained obscure in ESCC. The RARβ2 methylation status was assessed by methylation-specific PCR (MSP) in 12 ESCC cell lines and compared with their mRNA and protein expression level. Bisulfite sequencing of RARβ₂ promoter region was performed to confirm the MSP results. After 5-aza-2′-deoxycytidine (5-aza-dc) treatment the expression of RARβ₂ was reversed in two RARβ₂-downregulated cell lines. Therefore, hypermethylation of the promoter regions of RARβ2 gene is a major mechanism of transcriptional inactivation and might be involved in tumor development of esophagus in some ESCC cell lines suggesting that multiple mechanisms contribute to the loss of RARβ₂ expression in ESCC cell lines. Furthermore, the methylation status of RARβ₂ promoter region and its expression was analyzed in 51 ESCC tissue samples with their adjacent normal epithelia and two normal esophageal epithelia. The results showed that there was a statistically significant correlation between methylation status of RARβ₂ and tumor grade; Moreover, a relationship between methylation status and decreased RARβ₂ expression was found only in G₂ stage tumors. After 5-aza-dc treatment, RARβ₂ restoration was accompanied by growth inhibition and this might be one of the mechanisms but not the only mechanism for the tumor cell growth inhibition by 5-aza-dc. This study may have clinical applications for ESCC therapy and prevention.