Abstract Cancer cells employ multiple mechanisms to evade the recognition by the host immune system, including the expression of the negative T cell regulatory molecule PD-L1. PD-1/PD-L1-mediated immune escape plays key role during cancer development. It is crucial to unravel the regulatory mechanism of PD-1/PD-L1. In this study, we seek one enhancer (we named “enh9”) that regulates the transcription of PD-L1 by systematic exploration. First, we found that PD-L1 and enh9 were co-expressed in multiple cancer types, including both lung adenocarcinoma and lung squamous cell carcinoma, which was further validated in a cohort of 130 lung cancer cell lines from the Cancer Cell Line Encyclopedia database. Then the direct interaction between PD-L1 and enh9 was further confirmed by Hi-C dataset. To validate the transcriptional regulation of PD-L1 by enh9, we knocked out enh9 using CRISPRi (CRISPR/ clustered regularly interspaced short palindromic repeats interference) in lung adenocarcinoma cell line A549, and single cell clones with double-copy deletion of enh9 were screened out. The expression of PD-L1 was dramatically reduced at both mRNA and protein level in enh9 knockout cells. Furthermore, we found that the cell proliferation and metastasis of enh9 knockout cells were significantly decreased compared to the control cells. Based on the previous studies that PD-L1 intrinsically regulated cancer cells' proliferation in ovarian and melanoma cancer cells, ongoing work is to determine whether enh9 regulates the cancer cell hallmarks by direct regulation of the expression of PD-L1 in lung cancer cells. The regulatory mechanisms will be deeply studied. In conclusion, we found an enhancer regulated the expression of PD-L1, which was one famous immune checkpoint, and it may play key roles in the cancer cell hallmarks. Citation Format: Chunyan Li, Han Chen, Han Liang. One enhancer stimulates cancer cells proliferation and metastasis via the regulation of PDL1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 550.