Objective To investigate the expression of the immune checkpoint molecule programmed death ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC) and its regulatory relationship with the microRNA-200 (miRNA-200) family, and to elucidate the clinical significance and regulatory mechanism of PD-L1 in NSCLC. Methods NSCLC cancer tissues and matched paracancerous tissues (138 cases) were selected for this study. The PD-L1 expression was detected by immunohistochemistry and the expression levels of miRNA-200 family (miRNA-200a, miRNA-200b, miRNA-200c, miRNA-429, and miRNA-141) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Lung cancer A549 cells were cultured and transfected with five miRNA mimics of miRNA-200 family. The effect of miRNA-200 family mimics on PD-L1 expression and proliferation activity of A549 cells were detected by Western blotting and cell counting kit-8 (CCK-8) assay, respectively. Luciferase reporter gene assay was used to elucidate the regulation of PD-L1 by miRNA-200 family. Results Total positive rate of PD-L1 in 138 NSCLC tissues was 58.7% (81/138), which was significantly higher than that in paracancerous tissues (3.6%, 5/138) (P<0.05). The expression level of PD-L1 was not related to the patients' gender, age, tumor size or histological types, but was closely related to lymph node metastasis, vascular invasion and clinical TNM stage (all P<0.05). The miRNA-200 family was expressed at a low level in NSCLC tissues than that in paracancerous tissues (all P<0.01), and patients with positive PD-L1 expression had significantly lower levels of miRNA-200 family expression (all P<0.01). Overexpression of miRNA-200 family in A549 cells significantly down-regulated PD-L1 and inhibited proliferation activity of cancer cells (all P<0.01). Luciferase reporter gene assay confirmed that the miRNA-200 family directly and negatively regulated the expression of PD-L1 (all P<0.01). Conclusion High expression of PD-L1 in NSCLC indicates poor prognosis. PD-L1 is the target gene of miRNA-200 family, and the low expression of miRNA-200 family may be an important reason for the high expression of PD-L1 in NSCLC.