Background: Black pepper (Piper nigrum L.) is widely used as a traditional medicine, including usage for pain relief, fevers, as well as an anticancer agent. Previously, we reported that piperine-free P. nigrum extract (PFPE) inhibited breast cancer in vitro and in vivo. Objective: In this present study, we explored the anticancer effects of PFPE on cholangiocarcinoma (CCA). Materials and Methods: 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to analyze cytotoxic potential of PFPE whereas deoxyribonucleic acid (DNA) fragmentation followed by Western blot analysis were used. Results: PFPE composed of alkaloid, flavonoid, amide, lignans, opioid, and steroid. This crude extract represented cytotoxic effect against CCA cells which stronger than dichloromethane P. nigrum crude extract and piperine, especially on KKU-M213 (median inhibition concentration [IC50] at 13.70 μg/ml) and TFK-1 (IC50at 15.30 μg/ml). Interestingly, PFPE showed lower cytotoxicity against normal human cholangiocyte MMNK-1 cells (IC50at 19.65 μg/ml) than KKU-M213 and TFK-1 cells. Then, the molecular mechanisms of PFPE were firstly evaluated by DNA fragmentation followed by Western blot analysis. The degradation of DNA was observed on KKU-M213 and TFK-1 cells after treatment with PFPE at day 2. Then, proliferation proteins including topoisomerase II, AKT8 virus oncogene cellular homolog, avian myelocytomatosis virus oncogene cellular homolog, cyclin D1, signal transducer and activator of transcription 3, cyclooxygenase-2, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) were decreased and p21 was increased. Furthermore, apoptotic proteins, such as tumor protein p53, Bcl-2-associated X protein, and p53 upregulated modulator of apoptosis were upregulated. Meanwhile, antiapoptotic protein B-cell lymphoma 2 was down-regulated. Conclusion: These results indicated that PFPE inhibited CCA through the down-regulation of cell proliferation and induction of apoptosis pathway.