Programmed cell death-1(PD-1)/programmed cell death ligand-1(PD-L1)blocking ther-apy has become a major pillar of cancer immunotherapy.Compared with antibodies targeting,small-molecule checkpoint inhibitors which have favorable pharmacokinetics are urgently needed.Here we identified berberine(BBR),a proven anti-inflammation drug,as a negative regulator of PD-L1 from a set of traditional Chinese medicine(TCM)chemical monomers.BBR enhanced the sensi-tivity of tumour cells to co-cultured T-cells by decreasing the level of PD-Ll in cancer cells.In addi-tion,BBR exerted its antitumor effect in Lewis tumor xenograft mice through enhancing tumor-infiltrating T-cell immunity and attenuating the activation of immunosuppressive myeloid-derived sup-pressor cells(MDSCs)and regulatory T-cells(Tregs).BBR triggered PD-L1 degradation through ubi-quitin(Ub)/proteasome-dependent pathway.Remarkably,BBR selectively bound to the glutamic acid 76 of constitutive photomorphogenic-9 signalosome 5(CSN5)and inhibited PD-1/PD-Ll axis through its deubiquitination activity,resulting in ubiquitination and degradation of PD-L1.Our data reveals a previously unrecognized antitumor mechanism of BBR,suggesting BBR is small-molecule immune checkpoint inhibitor for cancer treatment.