Background Gastric cancer (GC) patients with PD-L1-negative tumor occasionally have a favorable response to anti-PD-1 mAb. The aim of the present study was to investigate the regulatory mechanism and immunosuppressive role of PD-L2 in GC. Methods We used immunohistochemistry to evaluate the expression of PD-L2 in primary tumors from 194 patients with GC. The mechanism of PD-L2 expression was assessed in TCGA stomach adenocarcinoma tissue dataset and in vitro assay using GC cell lines. The immunosuppressive role of PD-L2 was evaluated by cytotoxicity of CTL clone against PD-L2 expressing GC cells. Results PD-L2 was expressed on tumor cells (TCs) of 28.4% patients and PD-L2 expression on TCs was significantly associated with tumor progression. TCGA dataset revealed that IFN-γ and, to a lesser extent, IL-4 signature significantly correlated with PD-L2 expression. In vitro assay showed that IFN-γ and, also to a lesser extent, IL-4 can upregulate PD-L2 expression on GC cells. Anti-PD-L2 mAb significantly enhanced the cytotoxicity of CTL clone against GC cell lines expressing PD-L2. Conclusions PD-L2 is expressed on GC cells and PD-1/PD-L2 interaction are functionally involved in anti-tumor CTL activities. PD-L2 expression should be considered when determining the optimal immunotherapy for GC.