Although the inflammation‐associated cytokine interleukin‐6 (IL‐6) has been implicated in cholangiocarcinoma growth, the relationship between IL‐6 and oncogenic changes is unknown. IL‐6 can increase expression of DNA methyltransferase‐1 (DNMT‐1) and epigenetically regulate the expression of several genes, including microRNAs (miRNAs). DNMT‐1 up‐regulation occurs in hepatobiliary cancers and is associated with a poor prognosis. To understand the potential regulation of DNMT‐1 by IL‐6–dependent miRNAs, we examined the expression of a group of miRNAs which have sequence complementarity to the 3'‐untranslated region of DNMT‐1, namely miR‐148a, miR‐152, and miR‐301. The expression of these miRNAs was decreased in cholangiocarcinoma cells. Moreover, the expression of all three miRNAs was decreased in IL‐6–overexpressing malignant cholangiocytes in vitro and in tumor cell xenografts. There was a concomitant decrease in expression of the methylation‐sensitive tumor suppressor genes Rassf1a and p16INK4a. Using luciferase reporter constructs, DNMT‐1 was verified as a target for miR‐148a and miR‐152. Precursors to miR‐148a and miR‐152 decreased DNMT‐1 protein expression, increased Rassf1a and p16INK4a expression, and reduced cell proliferation. Conclusion: These data indicate that IL‐6 can regulate the activity of DNMT‐1 and expression of methylation‐dependent tumor suppressor genes by modulation of miR‐148a and miR‐152, and provide a link between this inflammation‐associated cytokine and oncogenesis in cholangiocarcinoma. (HEPATOLOGY 2010.)