In cancer immunology, the programmed cell death 1-programmed cell death 1/ligand 1 (PD-1/PD-L1) pathway plays a major role. Anti-PD-1 and anti-PD-L1 antibodies provide reliable immunotherapy when given as treatment for various types of malignancy including lung cancer. PD-L1 expression in cancer cells has been reported to be a predictive factor for the therapeutic effects of immunotherapy. However, the mechanism of PD-L1 expression remains unclear. Another key process in cancer progression is epithelial-mesenchymal transition (EMT). In the present study, we investigated the mechanism of PD-L1 expression as well as changes in its expression during the EMT process in non-small cell lung cancer (NSCLC). In this study, A549 cells underwent EMT by treatment with TGF-β or chemotherapeutic agents and then PD-L1 expression was evaluated. The alterations of PD-L1 expression was also examined during the reverse EMT process; mesenchymal-epithelial transition (MET). The relationship between for PD-L1 expression and EMT status in clinical specimens with NSCLC after induction chemotherapy were analyzed by immunohistochemical staining. We found that PD-L1 expression was upregulated following TGF-β induction; in contrast, it was downregulated by TGF-β receptor-kinase inhibitors and the MET process. Furthermore, chemo-treatment increased TGF-β expression and enhances PD-L1 expression via autocrine TGF-β induced EMT. Analysis of clinical samples revealed a significant relationship between PD-L1 expression and EMT status (P<0.05). In conclusion, our results suggest that PD-L1 expression is regulated by TGF-β induced EMT and enhanced by chemo-treatment via the chemo-induced TGF-β signaling. The anti-PD-1/PD-L1 blockade may provide more effective anticancer activities in combination with chemotherapy in NSCLC.