Objectives: Piper nigrum (PN) is well known for its cytotoxic and pharmacological benefits in several cancer cells. However, there is less documented evidence about its cytotoxic efficacy against cholangiocarcinoma (CCA). The roles of PN extract in two CCA cells, KKU-100, and KKU-M452, were evaluated. Materials and Methods: Viability was determined by sulforhodamine B and cell cycle distribution. Apoptotic effects were examined by flow cytometry after staining with Annexin V-FITC and Propidium idodide, JC-1, and Dichlorodihydrofluorescein (DCF-DA) staining. Migration was studied by Wound healing and Matrigel migration assay. Results: The results indicated that PN treatment significantly inhibited cells viability and cell replication by dose-and time-dependent both of two CCA cells. Growth was decreased by detecting the cell cycle arrest at G0/G1 phase in KKU-100 cells and S to G2/M phase in KKU-M452 cells. PN extract markedly induced cancer cells apoptosis after treatment for 24 h by loss of mitochondrial membrane potential and increasing of reactive oxygen species production. Furthermore, a significant reduction of migration was observed in both two cells, KKU-100 cells was suppressed the migratory ability more than KKU-M452 cells. Conclusion: The data demonstrated that PN extract directly suppresses proliferation and inhibits cells migration in two CCA cells. Finally, PN may be useful for CCA treatment.