Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor, which poses a serious threat to human health. Histone 3 lysine 9 trimethylation (H3K9me3) is a post-translational modification involved in regulating a broad range of biological processes and has been considered as potential therapeutic target in types of cancer. However, there is limited research on investigating profiles of histone modification H3K9me3 in ICC patients.In this study, we applied the ChIP-seq technique to investigate the effect of H3K9me3 on ICC. Anti-H3K9me3 antibody was used for ChIP-seq in ICC (RBE cell lines) and HIBEpic (normal cell lines). MACS2 (peak-calling tools) was then used to identify the peaks recorded in RBE and HIBEpic cell lines. Gene expression, mutation and clinical data were downloaded from TCGA and cBioPortal databases.H3K9me3 exhibited abnormal methylation and influenced the process of abnormal gene expression in patients suffering from ICC. The Wnt/β-Catenin signaling pathway (also known as simply the WNT signaling pathway) was enriched in H3K9me3-regulated genes.We are the first to report that H3K9me3 may play an important role in the progression of ICC. It promotes the understanding of epigenetic molecular mechanisms for ICC.© 2022. The Author(s).