This study investigated the genomic characteristics and immunotherapy efficacy of microsatellite instability-high (MSI-H) in cholangiocarcinoma (CCA). A total of 887 CCA patients were analyzed, with 5.4% (48/887) identified as MSI-H. MSI-H patients showed significantly higher tumor mutation burden (TMB) and PD-L1 expression compared to microsatellite stability (MSS) patients. Patients with MSI-H had longer overall survival (OS) and progression-free survival (PFS) when treated with PD-1 inhibitor-based immunotherapy. Integrating MSI-H status with PD-L1 expression (CPS ≥ 5) could distinguish immunotherapy efficacy. MSI-H was associated with higher TMB and more positive PD-L1 expression in CCA tumors. In advanced CCA patients receiving PD-1 inhibitor-based immunotherapy, MSI-H and positive PD-L1 expression were linked to improved OS and PFS. The study found that MSI-H status was a protective factor for both OS and PFS in multivariate analysis. However, PD-L1 expression was also a confounding factor. The TMB value of the MSI-H group was significantly higher than that of the MSS group, consistent with other studies. MSI-H patients had more indel alterations, similar to those in colorectal cancer. Mutated pathways such as SWI/SNF, BER, MMR, and HRR contributed to MSI-H status. The study also identified potential actionable targets (PATs) in CCA with different MSI statuses. The results suggest that MSI-H patients may benefit more from PD-1 inhibitor-based immunotherapy. The study highlights the importance of MSI-H status in CCA and its association with immunotherapy response. However, the study has limitations, including potential overestimation of MSI-H prevalence and the need for further validation in larger cohorts. The findings emphasize the role of MSI-H in CCA and its potential for precision medicine and immunotherapy.This study investigated the genomic characteristics and immunotherapy efficacy of microsatellite instability-high (MSI-H) in cholangiocarcinoma (CCA). A total of 887 CCA patients were analyzed, with 5.4% (48/887) identified as MSI-H. MSI-H patients showed significantly higher tumor mutation burden (TMB) and PD-L1 expression compared to microsatellite stability (MSS) patients. Patients with MSI-H had longer overall survival (OS) and progression-free survival (PFS) when treated with PD-1 inhibitor-based immunotherapy. Integrating MSI-H status with PD-L1 expression (CPS ≥ 5) could distinguish immunotherapy efficacy. MSI-H was associated with higher TMB and more positive PD-L1 expression in CCA tumors. In advanced CCA patients receiving PD-1 inhibitor-based immunotherapy, MSI-H and positive PD-L1 expression were linked to improved OS and PFS. The study found that MSI-H status was a protective factor for both OS and PFS in multivariate analysis. However, PD-L1 expression was also a confounding factor. The TMB value of the MSI-H group was significantly higher than that of the MSS group, consistent with other studies. MSI-H patients had more indel alterations, similar to those in colorectal cancer. Mutated pathways such as SWI/SNF, BER, MMR, and HRR contributed to MSI-H status. The study also identified potential actionable targets (PATs) in CCA with different MSI statuses. The results suggest that MSI-H patients may benefit more from PD-1 inhibitor-based immunotherapy. The study highlights the importance of MSI-H status in CCA and its association with immunotherapy response. However, the study has limitations, including potential overestimation of MSI-H prevalence and the need for further validation in larger cohorts. The findings emphasize the role of MSI-H in CCA and its potential for precision medicine and immunotherapy.