This study investigates the genomic characteristics and immunotherapy efficacy of microsatellite instability-high (MSI-H) cholangiocarcinoma (CCA). Among 887 CCA patients, 5.4% were identified as MSI-H, with a significantly higher tumor mutation burden (TMB) and more positive programmed death ligand 1 (PD-L1) expression compared to microsatellite stable (MSS) patients. In a cohort of 139 advanced CCA patients receiving PD-1 inhibitor-based immunotherapy, MSI-H patients had significantly longer overall survival (OS) and progression-free survival (PFS) compared to MSS patients. Integrating MSI-H with positive PD-L1 expression further distinguished the efficacy of immunotherapy. The study highlights the importance of MSI-H status and PD-L1 expression in predicting the response to immunotherapy in CCA, suggesting that MSI-H patients may benefit more from immunotherapy.This study investigates the genomic characteristics and immunotherapy efficacy of microsatellite instability-high (MSI-H) cholangiocarcinoma (CCA). Among 887 CCA patients, 5.4% were identified as MSI-H, with a significantly higher tumor mutation burden (TMB) and more positive programmed death ligand 1 (PD-L1) expression compared to microsatellite stable (MSS) patients. In a cohort of 139 advanced CCA patients receiving PD-1 inhibitor-based immunotherapy, MSI-H patients had significantly longer overall survival (OS) and progression-free survival (PFS) compared to MSS patients. Integrating MSI-H with positive PD-L1 expression further distinguished the efficacy of immunotherapy. The study highlights the importance of MSI-H status and PD-L1 expression in predicting the response to immunotherapy in CCA, suggesting that MSI-H patients may benefit more from immunotherapy.