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Objective To develop alternative medicine for reducing undesired side effects of chemotherapy in CCA patients, the anticancer activity of Tiliacora triandra leaf powder ethanolic (TLPE) extract against cholangiocarcinoma cell lines was investigated. Methods Antiproliferation was studied using the MTT assay while apoptosis induction and cell cycle arrest were analyzed by flow cytometry. The levels of key proteins and phenolic acid content were analyzed by western blotting and reversed-phase HPLC, respectively. Results TLPE extract inhibited CCA cell growth in a dose- and time-dependent manner, with IC50 values of 7.86 ± 0.05 µg/ml for KKU-M213B cells and 8.59 ± 0.36 µg/ml for KKU-100 cells at an exposure time of 72 h. TLPE extract inhibited the growth of CCA cell lines by inducing apoptosis of both cell lines and causing an increased population of KKU-100 cells at G0/G1 phase. TLPE extract up-regulated Ac-H3 but down-regulated p-ERK, p53, Bax, CDK4 and Bcl2 expressions in KKU-M213B cells. TLPE extract up-regulated Ac-H3, p21 and Bax but down-regulated p-ERK, p53, CDK4 and Bcl2 expressions in KKU-100 cells. Additionally, phenolic acids including p-hydroxybenzoic, vanillic, syringic, p-coumaric, ferulic and sinapinic acids were identified. Conclusion These results suggest the possibility of developing T. triandra leaf powder ethanolic extract as a chemotherapeutic or chemoprevention agent for cholangiocarcinoma.