Esophageal squamous cell carcinoma (ESCC) is an aggressive tumor, and it is urgently needed to develop novel therapeutic strategies including immunotherapy. Immunotherapy with inhibition of PD-1/PD-L1 interaction by therapeutic mAb would be a new promising treatment strategy for variety of cancer types including ESCC. In the present study, we investigated the up-regulation of the programmed death ligand 1 (PD-L1) due to epithelial-mesenchymal transition (EMT) in ESCC by IHC in the clinical tumor samples of both Tissue Microarray (TMA) samples (n = 177) and whole tissue samples (n = 21). Definition of EMT phenotype is as low E-cadherin expression, and for PD-L1, IHC was evaluated from intensity and proportion of membranous staining with or without cytoplasmic staining. As a result, the IHC stains of ESCC showed that PD-L1 expression on tumor cells was positively correlated with EMT status in TMA samples (p = 0.0004) and whole tissue samples (p = 0.0029). In conclusion, our in vivo study clearly demonstrated that PD-L1 expression was up-regulated in mesenchymal type tumors of ESCC. These findings provide a strong rationale for the clinical use of anti-PD-1/anti-PD-L1 monoclonal antibodies for advanced ESCC patients.