Background and aimCholangiocarcinoma (CCA) is usually diagnosed at a late stage, leading to treatment failure. Cannabidiol (CBD), exhibits diverse anti-cancer effects in various cancers, offering avenues for improving CCA treatment. This study investigated the effects of CBD on human CCA cells and the underlying mechanisms in vitro and in vivo. Experimental procedureThe effects of CBD on three CCA cell lines (KKU-213B, KKU-100, KKU-055) were assessed using the SRB assay, clonogenic assay, extent of cell-cycle arrest, and 3D holotomography. Morphological changes were examined using transmission electron microscopy, while mitochondrial function alterations (ROS levels and membrane potential) were studied using Mitosox, JC-1, and DCFH-DA. Cellular senescence induction was evaluated via SA-β-gal staining. Protein-associated autophagy and cellular senescence were analyzed using western blot and/or immunofluorescent assays. A xenograft model demonstrated the anti-tumor activity of CBD and the induction of cellular senescence through immunohistochemistry targeting PCNA, β-gal, and p21. Results and conclusionCBD effectively inhibited CCA cell proliferation, suppressed colony formation and induced G0/G1 phase cell-cycle arrest. Morphological examination revealed lipid droplets/vesicles in CCA cell lines. CBD induced autophagy by upregulating LC3BII, downregulating p62, and inhibiting the p-PI3K, p-AKT, and p-mTOR pathways. Additionally, CBD disrupted mitochondrial homeostasis by elevating ROS, reducing membrane potential, and induced cellular senescence by increasing the expression of p53 and p21. In-vitro results were confirmed by xenograft models. Overall, CBD suppresses proliferation and induces cell death, autophagy and senescence in CCA cells via the PI3K/AKT/mTOR pathway, which indicates a therapeutic option for CCA treatment.