Late-stage ovarian cancer (OC) has a poor prognosis and a high metastasis rate, but the underlying molecular mechanism is unclear. RNA binding proteins (RBPs) play important roles in posttranscriptional regulation in the contexts of neoplasia and tumor metastasis. In this study, we explored the molecular functions of a canonical RBP, Transformer 2β homolog (TRA2B), in cancer cells. TRA2B knockdown in HeLa cells and subsequent whole-transcriptome RNA sequencing (RNA-seq) analysis revealed the TRA2B-regulated alternative splicing (AS) profile. We disrupted TRA2B expression in epithelial OC cells and performed a series of experiments to confirm the resulting effects on OC cell proliferation, apoptosis and invasion. TRA2B-regulated AS was tightly associated with the mitotic cell cycle, apoptosis and several cancer pathways. Moreover, the expression of hundreds of genes was regulated by TRA2B, and these genes were enriched in the functions of cell proliferation, cell adhesion and angiogenesis, which are related to the malignant phenotype of OC. By integrating the alternatively spliced and differentially expressed genes, we found that AS events and gene expression were regulated independently. We then explored and validated the oncogenic functions of TRA2B by knocking down its expression in OC cells. The high TRA2B expression was associated with poor prognosis in patients with OC. In ovarian tissue, TRA2B expression showed a gradual increasing trend with increasing malignancy. We demonstrated the important roles of TRA2B in ovarian neoplasia and aggressive OC behaviors and identified the underlying molecular mechanisms, facilitating the targeted treatment of OC.© 2023 Zhou et al.