Background: Cholangiocarcinoma (CCA) exhibits poor response to the present chemotherapeutic agents and frequently develops drug resistance. Finding novel anticancer drugs might enhance patient outcomes. Tiliacorinine, a bisbenzylisoquinoline alkaloid from the Thai medicinal plant Tiliacora triandra, effectively induced apoptosis of human CCA cell lines and inhibited tumor growth in mice. Here, we elucidate further the molecular mechanisms underlining the cytotoxicity of tiliacorinine and its implication in overcoming gemcitabine-resistance of CCA cells.Methods: Cytotoxicity of tiliacorinine against CCA cell lines was assessed using MTT assay. The molecular signaling was determined using Western blot analysis. Molecular docking simulations were applied to predict the binding affinity and orientation of tiliacorinine to the possible binding site(s) of the target proteins.Results: Tiliacorinine induced apoptotic cell death of CCA cells in a dose-and time-dependent manner. Tiliacorinine significantly suppressed the expression of anti-apoptotic proteins, Bcl-xL and XIAP; activated apoptotic machinery proteins, caspase-3, caspase-9, and PARP; and decreased the levels of pAkt and pSTAT3. EGF/EGFR activation model and molecular docking simulations revealed EGFR, Akt, and STAT3 as potent targets of tiliacorinine. Molecular docking simulations indicated a strong binding affinity of tiliacorinine to the ATP-binding pockets of EGFR, PI3K, Akt, JAK2, and SH2 domain of STAT3. Tiliacorinine could synergize with gemcitabine and restore the cytotoxicity of gemcitabine against gemcitabine-resistant CCA cells.Conclusion: Tiliacorinine effectively induced apoptosis via binding and blocking the actions of EGFR, Akt, and STAT3.General signicance: Tiliacorinine is a novel multi-kinase inhibitor and possibly a potent anti-cancer agent, in cancers with high activation of EGFR.