Objective: To isolate from human cholangiocarcinoma QBC-939 cells, the tumor cells with stem cell properties and provide material for subsequent investigations on cholangiocarcinoma stem cells. Methods: By means of serum-free culture and fluorescence-activated cell sorting, the CD133+EpCAMhigh stem cell-like cells with stem cell properties were isolated from QBC-939 cells, and were continuously cultured in the serum-free medium for observing their sphere forming ability. Then, the colony formation rate, drug resistance and cell proliferation ability, and the expressions of stem cell-associated nuclear transcription factors that included OCT-4, Bmi-1 and E-cadherin, as well as the tumor formation ability after subcutaneous implantation in BALB/c mice were compared between CD133+EpCAMhigh stem cell-like cells and QBC-939 cells. Results: CD133+EpCAMhigh stem cell-like cells showed a relatively strong sphere forming ability in the serum-free medium. In CD133+EpCAMhigh stem cell-like cells compared with QBC-939 cells, the colony formation rate, resistance to lobaplatin, and proliferative ability were significantly increased; the OCT-4 and Bmi-1 protein expressions were up-regulated, while the E-cadherin protein expression was down-regulated; the rate of subcutaneous xenograft formation was significantly increased. All the differences had statistical significance (all P<0.05). Conclusion: The stem cell-like cells derived from cholangiocarcinoma QBC-939 cells possess stem cell properties, and can be used for cholangiocarcinoma stem cell research.