Background and Objective: Programmed Death-Ligand 1 (PD-L1) is a crucial immune checkpoint in gastric cancer (GC) and its expression can be palmitoylated. However, the effect of PD-L1 palmitoylation in GC remains unclear. This study aimed to investigate the role of ZDHHC3-mediated PD-L1 palmitoylation in GC chemoresistance and CD8+ T cell cytotoxicity. Materials and Methods: The expression levels of ZDHHC3 and PD-L1 in GC tissues were analyzed using public datasets. The interaction between ZDHHC3 and PD-L1 in GC cells (AGS) was investigated by co-immunoprecipitation and immunofluorescence. The effects of ZDHHC3 on PD-L1 stability, chemoresistance and CD8+ T cell cytotoxicity were examined in AGS and AGS/DDP cells using siRNA knockdown and overexpression strategies. Results: The ZDHHC3 and PD-L1 were found to be coordinately overexpressed in GC and ZDHHC3 directly interacted with and stabilized PD-L1 in GC cells. Overexpression of ZDHHC3 conferred chemoresistance to GC cells through PD-L1, while ZDHHC3 knockdown sensitized GC cells to chemotherapy. Furthermore, ZDHHC3-mediated PD-L1 stabilization suppressed CD8+ T cell cytotoxicity, which could be reversed by PD-L1 neutralization. Conclusion: The ZDHHC3-mediated PD-L1 palmitoylation contributes to chemoresistance and inhibits CD8+ T cell cytotoxicity in GC. Targeting the ZDHHC3-PD-L1 axis may be a promising strategy to overcome chemoresistance and enhance anti-tumor immunity in GC.