Uveal melanoma (UM) is the most common intraocular malignant tumor in adults. Due to the lack of effective treatments for metastatic UM, the survival of UM has not changed over the past 3 decades. Therefore, it is important to identify essential genes regulating the metastasis of UM.In this study, a genome-wide CRISPR knockout screen in an orthotopic mouse model of UM was performed to identify the regulatory genes conferring the metastatic phenotype. Loss-of-function analyses were performed to explore the function of G protein pathway suppressor 2 (GPS2) in UM metastasis in vitro and in vivo. RNA sequencing was performed to investigate the molecular mechanism underlying the function of GPS2 as a tumor suppressor in UM.Among the highest-ranking genes, we found several validated tumor suppressors, such as SHPRH, GPS2, PRPH2, and hsa-mir-1229; GPS2 was chosen as the candidate gene for further studies. GPS2 was lower expressed in the tumor tissues of UM patients. Furthermore, knocking-down GPS2 promoted the proliferation and metastatic abilities of UM cells both in vivo and in vitro. Finally, analysis of the transcriptome data revealed that silencing GPS2 upregulates oncogenic signaling pathways MAPK and PI3K-Akt, and in the meantime downregulates tumor suppressor signaling pathway Slit/Robo in UM cells.Altogether, our study proved that the GPS2 gene functions as a tumor suppressor and might be a novel potential therapeutic target for UM treatment.© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.