Cholangiocarcinoma (CCA) is a notoriously lethal tumor mostly due to the de novo or acquired resistance to traditional chemotherapy besides gemcitabine and, therefore, an increasing need for effective strategies to prevent and treat the poor prognosis of this tumor is required. Thymoquinone (TQ), a hopeful natural product derived from black cumin (Nigella sativa) has shown considerable antineoplastic properties. Whether TQ exerts antitumor effects on CCA cells in vitro and in vivo remains unknown. Examinations of cell viability assay, detection of cell cycle and apoptosis, electrophoretic mobility shift assay (EMSA), western blotting and immunohistochemistry were used in the present study. We demonstrated that TQ inhibited the growth of human CCA cell lines (TFK-1 and HuCCT1) in a dose- and time-dependent manner. Firstly, our results provided evidence that TQ not only inhibits the proliferation of CCA cells, induces cell cycle arrest and prompts cell apoptotic effect in vitro, but it also exhibits inhibitory effects of tumor growth and angiogenesis in vivo. The responsible mechanism is at least partially due to TQ inhibiting the growth of CCA cell lines induced by downregulation of PI3K/Akt and NF-κB and regulated gene products, including p-AKT, p65, XIAP, Bcl-2, COX-2, VEGF. Taken together, these results provide strong evidence of our hypothesis that TQ alone presents a promising therapeutic regimen for the treatment of CCA cells with better efficiency.