<p>The treatment landscape for advanced hepatocellular carcinoma (HCC) has changed significantly with the recent introduction of immunotherapy, particularly immune checkpoint inhibitors (ICIs), such as those for programmed cell death ligand 1 (PD-L1). However, the heterogeneous response to ICIs is still high, which may be related to different tumor microenvironment (TME) influenced by inflammatory cytokines like IL6. High expression of ADAM9 a type I transmembrane proteinases can affect tumor progression by cleaving various cell surface proteins PD-L1, and alter TME. Bufalin (BUF) a traditional Chinese medicine with important anti-cancer and anti-inflammation property has found to stimulates anti-tumor immune response by modulating TME, and enhances ICIs antitumor activity. In this study we first show the correlation between ADAM9 and HCC patient clinical outcomes and evaluate the stimulating effect of IL6 on ADAM9 expression and its protease activity mediates PD-L1 shedding, and investigates the immune modulating function of BUF to increases anti-PD-1 efficiency by inhibiting sPD-L1 induction in HCC cell lines and treated animals. We discovered that IL6 treatment can highly stimulate ADAM9 expression and its PD-L1 shedding activity, while BUF treatment down regulates ADAM9, and its protease activity by attenuating IL6. In conclusion BUF treatment can be considered as an adjuvant therapy lowering s PD-L1 formation to improve antitumor immunity and immunotherapy responses in HCC.</p> Graphical abstract <p></p>