Oxidative stress is a cause of inflammation–related diseases, including cholangiocarcinoma (CCA). Our previous studies in animal and human models indicated that oxidative stress is a major cause of CCA development. … Oxidative stress damages biomolecules leading to cell death. However, some cells can survive by adapting to oxidative stress conditions, and selective clonal expansion of these resistant cells would be involved in oxidative stress–related carcinogenesis. In the present study, we established a hydrogen peroxide (H2O2)-resistant cell line (ox-MMNK1-L) from a cholangiocyte cell line (MMNK1) by chronic treatment with 25 M H2O2. The ox-MMNK1-L cell line had a significantly higher cell proliferation and migration rates than the parental cells. Moreover, the ox-MMNK1-L cells showed decreased expression of early B cell factor 1 (EBF1) whereas the expression of EBF1 inhibitor (ZNF423) was increased compared to the parental cells. These findings suggest that EBF1 and ZNF423 are the oxidative stress-responsive genes in CCA. Therefore, the expression patterns of EBF1 and ZNF423 were detected in CCA tissues. CCA patients who had low EBF1 expression and high ZNF423 in the tumor tissues was related with poor prognosis. Roles of EBF1 and ZNF423 in CCA progression were further investigated in MMNK1 and CCA cell lines using specific siRNAs. EBF1-knockdown-MMNK1 cells have shown to increase stem-like cell property, cell migration activity and estrogen response whereas ZNF423-knockdowned CCA cells showed reduced proliferation activity compared with the control cells. Thus, oxidative stress induces tumorigenic properties via suppression of EBF1 expression and activation of ZNF423 expression, resulting in CCA progression with poor prognosis.