Cholangiocarcinoma (CCA) is a cancer arising from the neoplastic transformation of cholangiocytes. During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Zebularine (1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. In this study, we explore the effect and possible mechanism of action of zebularine on CCA cells. We demonstrate that zebularine exerts an antitumor effect on CCA cells. Zebularine treatment decreased the concentrations of DNA methyltransferase (DNMT) proteins, and DNMT1 knockdown led to apoptotic cell death in the CCA cell lines TFK-1 and HuCCT1. DNA methylation analysis demonstrated that zebularine induced DNA demethylation, and the GO Biological Process terms "hemophilic cell adhesion", "regulation of transcription, DNA-dependent" and "Wnt signaling pathway" were found to be significantly enriched in association with demethylated genes. Furthermore, we observed that zebularine treatment decreased β-catenin protein levels in TFK-1 and HuCCT1 cells. These results suggest that zebularine alters DNA methylation status, and that some aspect of DNA demethylation by zebularine induces suppression of the Wnt signaling pathway, which leads to apoptotic cell death in CCA. We previously reported a novel mechanism of zebularine-induced cell growth arrest and apoptosis in hepatocellular carcinoma via a DNA methylation-independent pathway. Together, our present and previous studies indicate that zebularine could function as both a DNMT inhibitor and a non-DNMT inhibitor reagent, and that, while the optimal usage of zebularine may depend on cancer type, zebularine may be useful for chemotherapy against cancer.