Background & Aims Intrahepatic cholangiocarcinoma (ICC) is the second most common liver malignancy. ICC typically features remarkable cellular heterogeneity and a dense stromal reaction. Therefore, a comprehensive understanding of cellular diversity and the interplay between malignant cells and niche cells is essential to elucidate the mechanisms driving ICC progression and to develop therapeutic approaches. Methods Herein, we performed single-cell RNA sequencing (scRNA-seq) analysis on unselected viable cells from 8 human ICCs and adjacent samples to elucidate the comprehensive transcriptomic landscape and intercellular communication network. Additionally, we applied a negative selection strategy to enrich fibroblast populations in 2 other ICC samples to investigate fibroblast diversity. The results of the analyses were validated using multiplex immunofluorescence staining, bulk transcriptomic datasets, and functional in vitro and in vivo experiments. Results We sequenced a total of 56,871 single cells derived from human ICC and adjacent tissues and identified diverse tumor, immune, and stromal cells. Malignant cells displayed a high degree of inter-tumor heterogeneity. Moreover, tumor-infiltrating CD4 regulatory T cells exhibited highly immunosuppressive characteristics. We identified 6 distinct fibroblast subsets, of which the majority were CD146-positive vascular cancer-associated fibroblasts (vCAFs), with highly expressed microvasculature signatures and high levels of interleukin (IL)-6. Functional assays indicated that IL-6 secreted by vCAFs induced significant epigenetic alterations in ICC cells, particularly upregulating enhancer of zeste homolog 2 (EZH2) and thereby enhancing malignancy. Furthermore, ICC cell-derived exosomal miR-9-5p elicited high expression of IL-6 in vCAFs to promote tumor progression. Conclusions Our single-cell transcriptomic dataset delineates the inter-tumor heterogeneity of human ICCs, underlining the importance of intercellular crosstalk between ICC cells and vCAFs,...