Programmed death ligand-1 (PD-L1) is an important immune checkpoint for cancer immunotherapy in clinic. In this study, we reported that platycodin D, a natural product isolated from an edible and medicinal plant Platycodon grandiflorus (Jacq.) A. DC., down-regulated the protein level of PD-L1 in lung cancer cells. Flow cytometry and immunofluorescence assay showed a weaker surface PD-L1 signal in NCI–H1975 cells after the incubation with platycodin D (10 μM) for 15 min compared to the control group. Jurkat T cells showed enhancive interleukin-2 secretion when co-cultured with platycodin D-treated NCI–H1975 cells, suggesting that platycodin D-induced PD-L1 reduction increases the activation of Jurkat T cells. An augmentation of PD-L1 protein was detected in the cell culture medium from platycodin D treatment group. Chlorpromazine (60 μM) almost abolished the platycodin D-mediated PD-L1 extracellular release and restored the membrane PD-L1. Finally, hemolysis assay exhibited that platycodin D-triggered PD-L1 extracellular release was independent of the hemolytic mechanism. Taken together, our study demonstrates that platycodin D reduces the protein level of PD-L1 in lung cancer cells via triggering its release into the cell culture medium, which sheds new light for the application of natural products in cancer immunotherapy.