Cholangiocarcinoma (CCA) is a highly heterogeneous group of malignant tumors with different molecular etiologies and clinical manifestations. Post-translational modifications (PTM) such as ubiquitination and phosphorylation are widely involved in the progression of CCA. Our aim was to elucidate the effect of the deubiquitinating enzyme OTU domain-containing protein 3 (OTUD3) on the molecular pathogenesis of CAA. OTUD3 and ARID3A exhibit direct binding interactions and cytosolic substructural co-localization in CCA cells.OTUD3 specifically removes the ARID3A proteins K240 and K329 OTUD3 specifically removes the ubiquitinated chains at K240 and K329 of ARID3A protein, thereby enhancing ARID3A stability. OTUD3 promotes CCA proliferation and metastasis in vivo and in vitro by interacting with ARID3A.GSK3 beta interacts with OTUD3 protein and mediates phosphorylation of the Ser9 site of OTUD3, which enhances the affinity of OTUD3 to ARID3A and further stabilizes ARID3A protein. The expression of OTUD3 and ARID3A in CCA tissues is highly correlated, and the high expression of both proteins is closely related to the poor prognosis of patients. In conclusion, GSK3 beta phosphorylates OTUD3, enhances its binding ability to ARID3A, and ultimately inhibits the ubiquitination degradation of ARID3A, which promotes the progression of CCA. The GSK3 beta-OTUD3-ARID3A signaling pathway has been demonstrated for the first time in the progression of CCA.