Whether promoter hypermethylation of phosphatase and tensin homologue deleted from chromosome 10 (PTEN) is associated with loss of PTEN expression was not yet elucidated in esophageal squamous cell carcinoma (ESCC). The methylation status of PTEN gene was evaluated in 74 ESCC specimens and four esophageal cancer cell lines. Its association with clinicopathological factors or the prognosis was investigated by statistical analysis. We further measured messenger RNA (mRNA) and protein level of PTEN by quantitative RT-PCR and immunohistochemistry and studied the role of PTEN hypermethylation in loss of PTEN expression in clinical samples. Next, demethylation of PTEN gene with 5-azaC in EC9706 was performed to confirm the clinical findings. PTEN methylation was only found in 14 (18.9 %) of 74 ESCC tumor samples and one (EC9706) of four esophageal cancer cell lines. PTEN methylation was not statistically associated with clinicopathological factors and the prognosis (p > 0.05). In addition, 41 patients (55.4 %) and 38 patients (51.4 %) showed reduced mRNA level of PTEN and negative expression of PTEN protein in ESCC tumors, respectively. Detailed analysis indicated that PTEN methylation was a possible mechanism of loss of PTEN expression in ESCC, and further 5-azaC demethylation revealed inversed methylation status and increased mRNA or protein level of PTEN in EC9706. However, the role of PTEN methylation in loss of PTEN expression was still limited due to low frequency of methylation in ESCC. PTEN hypermethylation is a rare event and did not play an important role in the prognosis and loss of PTEN expression in ESCC.