Cholangiocarcinoma (CCA) is a rare type of liver cancer with a very poor prognosis. The prevalence of CCA is markedly variable with the highest incidence in the northeast Thailand, followed by other parts of Southeast Asia and China. Currently, there is still no reliable biomarker for diagnosis or treatment. NADPH-quinone oxidoreductase 1 (NQO1) is a xenobiotic metabolizing enzyme detoxifying chemical stressors and antioxidants, thereby providing cytoprotection in normal tissues. However, NQO1 is over-expressed in some cancers, suggesting roles in carcinogenesis and tumor progression. In this study, we examined NQO1 activity in surgical specimens from CCA patients and found much higher values than in the adjacent normal tissues. Immunohistochemical analysis revealed strong staining in tumor epithelial elements, whereas the non-tumor bile ducts and liver parenchyma were weakly stained. NQO1 mRNA expression in tumor tissues was widely varied among 43 patients. A significant association was observed between high level of NQO1 expression and short overall survival time by the Cox proportional hazard ratio of 2.40, p<0.05. By histological classification, non-papillary adenocarcinoma was an independent predictor for poor prognosis with the hazard ratio of 2.79, p<0.05. NQO1 expression may serve as a prognostic biomarker for the CCA.