<p>Human cytomegalovirus (HCMV) is associated with bile duct disorders in immunocompetent and immunocompromised individuals, including sclerosing cholangitis, biliary structures, AIDS cholangiopathy, biliary atresia (BA), and post-transplant biliary complications. Despite these clinical associations, it remains unknown whether HCMV can directly infect the bile duct epithelium and cause pathogenesis. Here, we establish a human iPSC-derived cholangiocyte-like cell (CLC) organoid model that can be infected by HCMV, which results in reduced organoid growth, a deformed structure, and a loss of barrier function. Bulk RNA sequencing (RNA-seq) showed that the HCMV-infected organoids had highly enriched expression of genes involved in the epithelial–mesenchymal transition (EMT) pathway. Importantly, blockade of TGF-β signalling abrogates the EMT induction effect in the HCMV-infected CLC organoids. Furthermore, single-cell RNA-seq indicates that HCMV infection occurred in different clusters with distinct viral and host gene expression profiles and the analysis shows increased expression of EMT and TGF-β signalling-related genes. The findings are confirmed in BA patient liver tissues by examining HCMV-DNA⁺ cells. Two modes of infection by HCMV are found in these CLC organoids, which show productive and latency-like features. This study shows that HCMV infection induces EMT in cholangiocytes that may contribute to the cholangiopathy seen in various diseases.</p>