The effects of treatments to programmed death ligand-1 (PD-L1) expression is unknown. The aim of this study was to investigate the impact of neoadjuvant chemotherapy (NACT) on PD-L1 expression in non-small cell lung cancer (NSCLC) patients. PD-L1 expression was detected by immunohistochemistry (IHC) method in 32 paired tumor specimens pre and post-NACT. The positivity of PD-L1 on tumor cells (TCs) changed from 75% to 37.5% after NACT (p = 0.003). Cases with IHC score of 1, 2, 3 all underwent apparent decrease (p = 0.007). However, no significant changes were observed on tumour-infiltrating immune cells (ICs) (p = 0.337). Subgroup and semiquantitative analyses all presented similar results. Moreover, patients with response to NACT presented significantly reduced PD-L1 expression on TCs (p = 0.004). Although it was not confirmed by the Cox proportional hazard regression model, there was an apparent difference in disease-free-survival (DFS) between negative-to-positive switch of PD-L1 status and the contrary group (median DFS: 9.6 versus 25.9, p = 0.005). Our data revealed that antecedent chemotherapy for NSCLC may results in inconsistency of PD-L1 expression. PD-L1 expression is suggested to be monitored around treatment and on serial samples, at least, on the latest tumor specimen.