Background: Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with poor prognosis. Among RNA modifications that drive tumor progression, N4-acetylcytidine (ac4C) mediated by NAT10 is crucial for RNA stability and translation. Cordycepin, a bioactive compound from Cordyceps sinensis, has known antitumor activity, yet its impact on ICC via RNA-modification pathways remains unclear. This study examines how cordycepin targets the NAT10-ac4C axis to inhibit ICC, providing mechanistic support for RNA-modification-based therapy. Methods: The antiproliferative effects of cordycepin on HUCCT1 and RBE cells were quantified using CCK-8 assays to determine IC 50 values. Western blotting was employed to profile RNA-modifying enzymes and identify ac4C/NAT10 as potential targets. The effects of cordycepin on cellular phenotypes were systematically assessed via CCK-8, EdU incorporation, colony formation, flow cytometry, Transwell migration and invasion, and wound-healing assays. Direct cordycepin-NAT10 interaction was validated by molecular docking, cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) analysis. Global ac4C levels were examined by dot blotting. RNA-seq combined with acRIP-seq was used to define downstream ac4C-regulated genes. Antitumor efficacy was further evaluated in C57BL/6 mouse subcutaneous ICC xenograft models. Results: Cordycepin exhibited IC 50 values of 67.18 µM in HUCCT1 cells and 75.50 µM in RBE cells. It markedly suppressed proliferation, migration, and invasion while inducing apoptosis in a dose-dependent manner (P < 0.05). Cordycepin significantly reduced NAT10 protein abundance and global ac4C modification levels (P < 0.05). Docking analysis predicted a binding energy of −7.0 kcal/mol, and CETSA together with DARTS confirmed direct target engagement. Integrative RNA-seq and acRIP-seq analyses identified VCAN, an extracellular matrix-associated gene, as a principal ac4C-regulated downstream effector. Cordycepin decreased both ac4C enrichment and expression of VCAN, whereas NAT10 overexpression reversed these effects. In vivo, cordycepin substantially suppressed tumor growth, reflected by reduced tumor volume and weight (P < 0.05). Correspondingly, NAT10 and VCAN expression in tumor tissues were significantly downregulated. Conclusion: This study demonstrates that cordycepin directly targets NAT10, attenuating its expression and global ac4C modification, thereby suppressing the downstream oncogenic effector VCAN and restraining ICC progression. These findings underscore the central role of the NAT10-ac4C axis in cordycepin's antitumor activity and identify this pathway as a promising therapeutic target in ICC.