Objective ·To comprehensively analyze the role, prognosis, and immune infiltration characteristics of splicing factor 3B subunit 1 (SF3B1) mutations in uveal melanoma (UVM). Methods ·A total of 20 patients diagnosed with primary UVM who underwent enucleation at Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, between 2018 and 2021 were included. DNA and RNA were extracted from tumor tissue samples and used for Sanger sequencing and RNA sequencing, respectively. In addition, gene expression data, mutation information, and clinical data from 80 UVM patients were obtained from The Cancer Genome Atlas (TCGA) database. Raw gene expression data (STAR-counts) were converted into transcripts per million (TPM) and subsequently normalized using log₂(TPM+1) for downstream analyses. According to SF3B1 mutation status, the TCGA-UVM cohort was divided into the SF3B1 mutant group and the SF3B1 wild-type group. The mutation frequency of SF3B1 was compared between the local cohort and the TCGA-UVM cohort, and Kaplan-Meier survival analysis was performed to evaluate patient prognosis in the TCGA-UVM cohort. Gene sets of common oncogenic pathways were obtained from the Molecular Signatures Database (MSigDB), and single-sample gene set enrichment analysis (ssGSEA) was used to evaluate the activity of relevant biological pathways in the TCGA-UVM cohort. Differential expression analysis was conducted using the R package limma, followed by functional enrichment and signaling pathway analyses using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Immune cell infiltration levels in the TCGA-UVM cohort were evaluated using the Tumor Immune Estimation Resource (TIMER) database. Combined with transcriptomic data of immune checkpoint-related genes, such as programmed cell death protein 1 (PDCD1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4), the association between SF3B1 mutation status and tumor immune microenvironment characteristics was further analyzed. The Tumor Immune Dysfunction and Exclusion (TIDE) algorithm was applied to predict the impact of SF3B1 mutation on responsiveness to immune checkpoint inhibitor therapy. Alternative splicing (AS) events were analyzed based on RNA-sequencing data from local cohort to identify SF3B1 mutation-associated differential AS events and transcript isoform alterations. Results ·Comparison between the local cohort and the TCGA-UVM cohort showed that the mutation frequency of SF3B1 in Chinese patients was markedly higher than that in the TCGA-UVM cohort. Survival analysis indicated that patients in the SF3B1 mutant group had significantly better overall survival and disease-specific survival than those in the SF3B1 wild-type group (both P<0.05). ssGSEA analysis indicated that SF3B1 mutation was associated with increased activities of several tumor-related pathways, including inflammatory response, hypoxia response, epithelial-mesenchymal transition, and angiogenesis. GO and KEGG analyses indicated that differentially expressed genes were primarily enriched in nuclear receptor activity and the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Immune infiltration analysis showed that SF3B1 mutation was associated with increased B-cell infiltration and lower expression of immune checkpoint-related genes (P<0.05). The TIDE algorithm suggested that SF3B1 mutation was associated with a higher response rate to immunotherapy (P<0.05). AS analysis further revealed that SF3B1 mutation was associated with multiple differential alternative splicing events and transcript isoform changes. Conclusion ·SF3B1 mutation occurs at a relatively high frequency in Chinese patients with UVM, and is associated with favorable survival outcomes and distinct immune infiltration characteristics, suggesting its potential as a biomarker for predicting prognosis and immunotherapy response in UVM.