Simple SummaryThis study investigated the antiproliferative activity of Krukovine (KV) in oxaliplatin-resistant pancreatic cancer cells and explored the mechanism of action. KV suppresses tumor progression via the downregulation of the Erk-RPS6K-TMEM139 signaling pathway in oxaliplatin-resistant pancreatic cancer cells. Transmembrane protein 139 (TMEM139) has been identified as a novel oncogene and its overexpression has been associated with various types of cancer, including pancreatic cancer. In this study, KV significantly downregulated TMEM139 expression in oxaliplatin-resistant pancreatic cancer cells. The downregulation of TMEM139 by KV may contribute to its antiproliferative activity and could be a potential target for future therapeutic interventions. KV could be a potential therapeutic agent for the treatment of pancreatic cancer, particularly for patients withKrasmutations and oxaliplatin-resistant tumors.AbstractKrukovine (KV) is an alkaloid isolated from the bark ofAbuta grandifolia(Mart.) Sandw. (Menispermaceae) with anticancer potential in some cancers withKRASmutations. In this study, we explored the anticancer efficacy and mechanism of KV in oxaliplatin-resistant pancreatic cancer cells and patient-derived pancreatic cancer organoids (PDPCOs) withKRASmutation. After treatment with KV, mRNA and protein levels were determined by RNA-seq and Western blotting, respectively. Cell proliferation, migration, and invasion were measured by MTT, scratch wound healing assay, and transwell analysis, respectively. Patient-derived pancreatic cancer organoids (PDPCOs) withKRASmutations were treated with KV, oxaliplatin (OXA), and a combination of KV and OXA. KV suppresses tumor progression via the downregulation of the Erk-RPS6K-TMEM139 and PI3K-Akt-mTOR pathways in oxaliplatin-resistant AsPC-1 cells. Furthermore, KV showed an antiproliferative effect in PDPCOs, and the combination of OXA and KV inhibited PDPCO growth more effectively than either drug alone.