Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, requiring novel treatments to target both cancer cells and cancer stem cells (CSCs). Altered splicing is emerging as both a novel cancer hallmark and an attractive therapeutic target. The core splicing factor SF3B1 is heavily altered in cancer and can be inhibited by Pladienolide-B, but its actionability in PDAC is unknown. We explored the presence and role of SF3B1 in PDAC and interrogated its potential as an actionable target. Methods SF3B1 was analyzed in PDAC tissues, an RNA-seq dataset, and publicly available databases, examining associations with splicing alterations and key features/genes. Functional assays in PDAC cell lines and PDX-derived CSCs served to test Pladienolide-B treatment effects in vitro, and in vivo in zebrafish and mice. Results SF3B1 was overexpressed in human PDAC and associated with tumor grade and lymph-node involvement. SF3B1 levels closely associated with distinct splicing event profiles and expression of key PDAC players (KRAS, TP53). In PDAC cells, Pladienolide-B increased apoptosis and decreased multiple tumor-related features, including cell proliferation, migration, and colony/sphere formation, altering AKT and JNK signaling, and favoring proapoptotic splicing variants (BCL-XS/BCL-XL, KRASa/KRAS, Δ133TP53/TP53). Importantly, Pladienolide-B similarly impaired CSCs, reducing their stemness capacity and increasing their sensitivity to chemotherapy. Pladienolide-B also reduced PDAC/CSCs xenograft tumor growth in vivo in zebrafish and in mice. Conclusion SF3B1 overexpression represents a therapeutic vulnerability in PDAC, as altered splicing can be targeted with Pladienolide-B both in cancer cells and CSCs, paving the way for novel therapies for this lethal cancer.