<p>Gastric cancer (GC) is a highly malignant tumor with significant morbidity and mortality rates globally. Recent studies have shown that RUNX2, a member of the RUNX family known for its role in osteoblast differentiation and bone morphogenesis, is associated with the pathogenesis and progression of GC, while the underlying mechanisms of the pathogenesis and progression of GC are largely unknown. In this study, we investigated the role of RUNX2 in GC progression by analyzing its effects on gene expression and alternative splicing (AS) in HGC-27 cells. We silenced RUNX2 by small interfering RNA (siRUNX2), and then analyzed the globally regulated transcriptome profile by sequencing method (RNA-seq) to identify the differentially expressed genes (DEGs) and alternative splicing (AS) genes. The downstream targets of RUNX2 were identified by performing CUT&amp;Tag and sequencing experiment in HGC-27 cells. Using RNA-seq, we identified 314 DEGs and 1120 regulated AS events (RASEs) in HGC-27 cells upon RUNX2 knockdown. The DEGs were enriched in pathways related to cell cycle regulation and apoptosis, while the RASEs were predominantly involved in cell cycle processes. These findings suggest that RUNX2 not only modulates gene expression but also extensively influences AS, which is crucial for cellular processes such as proliferation and survival. Notably, we found that RUNX2 regulates the expression of the splicing factor SF3B6 by binding to its promoter region. Our results showed that SF3B6 expression was significantly decreased in siRUNX2 samples, and its downregulation was associated with altered AS profiles of genes involved in the cell cycle. Our findings demonstrate that RUNX2 modulates the transcriptome and AS profile in GC cells, with a particular focus on its regulation of SF3B6. This study highlights the multifaceted role of RUNX2 in GC progression and suggests that targeting the RUNX2-SF3B6 axis could be a promising therapeutic strategy for GC.</p>