The expression of RSPO3 was determined by the balance between the DNA methyltransferase DNMT3a and the DNA demethylase TET1 in CCA. In this study, we conducted in vitro and in vivo experiments to investigate the effects of targeted RSPO3 promoter DNA methylation and demethylation on the tumorigenicity of CCA. This evidence concerns the gene DNMT3A and cholangiocarcinoma.