It has been reported that inhibition of GPR65 may be effective for the treatment of certain cancers. Nevertheless, the role of GPR65 in various cancers remains unknown. We conducted an exhaustive pan-cancer analysis of GPR65 using multiple databases, including TCGA, GTEx, BioGPS, HPA, cBioPortal, and GeneCards. GPR65 was found to be differentially expressed in various cancers and linked to tumor mutational burden (TMB), microsatellite instability (MSI), and Ploidy, playing a key function in the tumor microenvironment (TME). It is closely linked to the development of Th17 cells as well as Th1 and Th2 cells in certain cancers. Our findings indicate that the expression of GPR65 is highly linked with clinical prognosis, mutations, and immune cell infiltration. It was revealed as an indicator of patient prognosis as well as a possible immunomodulatory role. As a possible new immunological checkpoint, GPR65 could be a target for tumor immunotherapy.