Background <p>Dysregulation of alternative splicing (AS) has been identified as a promising target for cancer therapy. Nevertheless, the precise molecular mechanisms by which AS influences ovarian cancer (OC) progression have not yet been fully elucidated.</p> Methods <p>A comprehensive bioinformatics analysis was conducted to identify and screen core splicing factors in OC. The splicing factor EFTUD2 was found to be significantly overexpressed in clinical OC samples. Subsequent in vitro and in vivo assays elucidated the oncogenic role of EFTUD2 in OC. RNA-seq and AS events analysis were employed to determine the key downstream target regulated by EFTUD2. ASOs targeting EFTUD2 were developed for efficacy validation.</p> Results <p>EFTUD2 was identified as a critical splicing factor in the pathogenesis of OC, and EFTUD2 knockdown impeded OC tumorigenesis and progression. The EFTUD2-ASO significantly inhibited tumor growth in vivo. Mechanistically, EFTUD2 was shown to promote the malignant biological behavior of OC by facilitating the efficient splicing of DDX41 and maintaining the oncogenic expression of its functional proteins. Knockdown of DDX41 partially mitigated the EFTUD2-induced malignant progression of OC cells.</p> Conclusions <p>Our findings suggest that the EFTUD2/DDX41 axis is a viable target for OC. ASO-mediated silencing of EFTUD2 presents promising new therapeutic options for OC patients.</p>