Abstract: Objective To explore the association of PTEN gene hypermethylation status and the occurrence and development of esophageal squamous cell cancer (ESCC), and the relationship of PTEN gene hypermethylation with mRNA expression. Methods Firstly, PTEN methylated pattern was determined by methylation specific polymerase chain-reaction (MSP) among 94 ESCC patients. In the meantime, the relationship between PTEN methylated pattern and the risk of ESCC, lymphatic metastasis, penetration, pTNM staging were analyzed. Secondly, PTEN mRNA expression was detected by reverse transcription PCR (RT-PCR), and the relationship between PTEN genes methylation and mRNA expression was analyzed. Results The frequency of PTEN gene hypermethylation among the 94 tumor tissues was 45.7%(43/94), while that in adjacent normal tissues was 11.7% (11/94), with significant difference (P=0.00). The frequency of PTEN hypermethylation in the positive lymphatic metastasis group (62.9%, 22/35) was significantly higher than that in negative lymphatic metastasis group (35.6%, 21/59, P=0.01). No significant difference were observed for the frequency of PTEN hypermethylation between two groups with different depth of penetration (P=0.23) or different pTNM staging (P=0.14). Among the tumor tissues with negative mRNA expression of PTEN, 74.2% (23/31) cases showed PTEN hypermethylation. The lost mRNA expression of PTEN was significantly associated with PTEN hypermethylation (Phi=-0.40, P＝0.00). Conclusion The high frequency of PTEN hypermethylation was associated with the development and lymphatic metastasis of ESCC. In addition, PTEN hypermethylation is one of the reasons for PTEN inactivation.