Background: Programmed death-ligand 1 (PD-L1), which can be induced by interferon-gamma (IFN-γ) in the tumor microenvironment, is a critical immune checkpoint in cancer immunotherapy. Natural products which reduce IFN-γ-induced PD-L1 might be exert immunotherapy effect. Licochalcone A (LCA), a natural compound derived from the root of Glycyrrhiza inflata Batalin. (Fabaceae), was found to interfere IFN-γ-induced PD-L1. Purpose: The aim of this study is to further clarify the effect and the mechanism of LCA on inhibiting IFN-γ-induced PD-L1 in lung cancer cells. Methods: The expression levels of PD-L1 were evaluated by flow cytometry, western blot and qRT-PCR. Click-iT protein synthesis assay and luciferase assay were used to identify the effect of LCA on protein synthesis. Jurkat T cell proliferation and apoptosis in the co-culture system were detected by flow cytometry. Flow cytometry was also applied to evaluate reactive oxygen species (ROS) generation. Results: LCA downregulated IFN-γ-induced PD-L1 protein expression and membrane localization in human lung cancer cells, regardless of inhibiting PD-L1 mRNA level or promoting its protein degradation. LCA decreased apoptosis and proliferative inhibition of Jurkat T cells caused by IFN-γ-induced PD-L1-expressing in A549 cells in the co-culture system. Strikingly, LCA was verified as a protein synthesis inhibitor, which reduced both cap-dependent and -independent translation. LCA inhibited PD-L1 translation, likely due to inhibition of 4EBP1 phosphorylation (Ser 65) and activation of PERK-eIF2α pathway. Furthermore, LCA induced ROS generation in a time-dependent manner in lung cancer cells. N-acetyl-L-cysteine (NAC) not only revered ROS generation triggered by LCA but also restored IFN-γ-induced expression of PD-L1. Both the inhibition of 4EBP1 phosphorylation (Ser 65) and activation of PERK-eIF2α axis triggered by LCA was restored by co-treatment with NAC. Conclusion: LCA abrogated IFN-γ-induced PD-L1 expression via ROS generation to abolish the protein translation, indicating that LCA has the potential to be applied in cancer immunotherapy.