Objective: To investigate the cytotoxic, apoptotic and inhibitory activities on cell migration and invasion of plumbagin in the human cholangiocarcinoma (CCA) cell line (CL-6) in comparison with human embryonic fibroblast cell line (OUMS). Methods: Cytotoxicity activity was evaluated using MTT assay. Inhibitory effect on cell migration and invasion were investigated using label-free real-time cell analysis and QCM ECMatrix cell invasion chamber, respectively. Apoptotic activity was evaluated using flow cytometry and CellEvent? Caspase 3/7 assay. Results: Based on results of the cytotoxicity test in CL-6 cells, 50％ inhibitory concentration (IC50, Mean±SD) values of plumbagin and the standard drug 5-fluorouracil were (24.00±3.33) and (1036.00±137.77) μmol/L, respectively. The corresponding values for OUMS cells were (57.00±5.23) and (2147.00±209.98) μmol/L, respectively. The selectivity index was 2.28. The inhibitory activities of plumbagin on cell migration and invasion were potent and concentration-dependent with IC50 of 25.0 μmol/L and complete inhibition at 25.0μmol/L. Flow cytometry analysis showed that plumbagin at 12.5 μmol/L (half IC50) induced CL-6 cell apoptosis (43.24％ of control) through stimulation of caspase 3/7 activities. Complete cell apoptosis was observed at 12.5 μmol/L. Conclusions: The cytotoxic activity and inhibition of migration and invasion including apoptosis induction in the human CCA cell line (CL-6) suggest that plumbagin could be a promising candidate for CCA chemotherapeutics. However, its relatively low selective cytotoxic effect on CCA cells is a major conc.