Resistance to chemotherapy is the major problem in cancer treatment. Cholangiocarcinoma (CCA) is the tumor arising from the bile duct epithelium. The disease is characterized by very poor prognosis and rarely responds to current radiotherapy or chemotherapy. Transcription factor Nrf2 is activated by oxidative stress and electrophiles and contributes to cytoprotection in normal cells as well as cancer cells. Inhibition of Nrf2 can enhance the sensitivity of cancer cells to chemotherapeutic agents, although this sensitizing effect is variable depending on the cancers. In this study, we selected three CCA cell lines with different Nrf2 expression levels, detected by immunocytofluorescent staining. Chemotherapeutic agents variably induced the expression of antioxidant and xenobiotic metabolizing genes including Nrf2, NQO1, HO-1, GCLC, and GSTP1. Knockdown of Nrf2 expression by siRNA suppressed protein expression of Nrf2-regulated genes and enhanced the sensitivity to 5-fluorouracil and gemcitabine of CCA cells in both high and low basal Nrf2 expression. Cells with more resistance to chemotherapeutic agents gained more chemosensitizing effect by Nrf2 inhibition than the sensitive cells. The IC50 of the chemotherapeutic agents was also significantly reduced and the maximal cytotoxic effect was increased. Suppression of Nrf2 signaling may be a strategy to increase the efficacy of chemotherapy to CCA.