The poor prognosis of esophageal squamous cell carcinoma (ESCC) is mainly attributed to higher lymphatic-related metastatic ability. Whether the loss of expression of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is associated with lymphatic-related metastasis needs elucidation. In the present study, we assessed the mRNA and protein level of PTEN in ESCC by qRT-PCR and immunohistochemistry. The results showed PTEN mRNA level in tumors was significantly lower than that in corresponding non-tumor esophageal epitheliums (p<0.001), while 38 (51.4%) tumor samples were negative for expression of PTEN in ESCC tumors. Then the association between negative expression of PTEN and lymphatic-related metastasis (lymph node metastasis/3-year postoperative lymphatic metastatic recurrence) was evaluated. The proportion of PTEN-negative expression was significantly higher in positive lymph node metastasis (pN+) than that in negative lymph node metastasis (pN0) (p=0.021). The negative expression of PTEN was not an independent risk factor for the lymphatic recurrence rate in multivariate analysis (p=0.498), however, the lymphatic recurrence rate (60.5%) in PTEN-negative expression group was higher than that (36.1%) in PTEN-positive expression group (p=0.019). Furthermore, PTEN expression was stably silenced by lentiviral-vectored shRNA (Lenti-shRNA) in Eca109 (ESCC-derived cell line) to study functional effect of PTEN in vitro and in vivo. The laboratory study indicated increased cell proliferation, migration and invasion in vitro and more rapid growth rate of xenograft tumors in vivo after stable silencing of PTEN expression. Moreover, we proved that FAK/pFAK were not the main factors mediating the mechanism of metastasis in ESCC. In conclusion, negative expression of PTEN could be a useful biomarker to predict high risk for lymphatic-related metastasis in ESCC.