Gastric cancer (GC) is one of the most typical cancerous neoplasms occurring in the digestive system. For advanced GC, immunotherapy is the final option for them to prolong survival time. Hence, we aimed to identify new molecular targets to enhance the immunotherapy response in GC individuals. Then we applied bioinformatic analysis to explore the expression profiles of G-protein-coupled receptor 27 (GPR27) transcription and GPR27 methylation. The associations between survival of GC patients and GPR27 transcription and methylation were then analyzed. We also studied the link between GPR27 expression and levels of immune cell infiltration. Finally, we gained insights into the prognostic role of GPR27 protein in 97 cases of GC individuals. According to datasets gained from TCGA, GPR27 mRNA is expressed lower in GC tissues. Down-regulation of GPR27 transcription was related with better survival in GC individuals, and GPR27 cg03024619 had the most significant prognostic value (HR=0.553, P<0.0001). In addition, the expression level of GPR27 has a clear interaction with immune cells' infiltration and their markers. Single-cell analysis displayed that GPR27 is mainly expressed in macrophages. Finally, down-regulation of GPR27 protein was observed in GC tissues and correlated with better survival outcomes. GPR27 can serve as an important prognostic biomarker and exert an immunomodulatory role in GC. Our findings highlight the significance of GPR27 in a variety of cancers, including GC, and provide clues for a better understanding of GPR27 from bioinformatics and clinically validated perspective.