BACKGROUND: The association between gut bacteria and the response to immune checkpoint inhibitors (ICI) in hepatocellular carcinoma (HCC) has been studied; however, multi-kingdom gut microbiome alterations and interactions in ICI-treated HCC cohorts are not fully understood. METHODS: From November 2018 to April 2022, patients receiving ICI treatment for advanced HCC were prospectively enrolled. Herein, we investigated the multi-kingdom microbiota characterization of the gut microbiome, mycobiome, and metabolome using metagenomic, ITS2, and metabolomic data sets of 80 patients with ICI-treated HCC. RESULTS: Our findings demonstrated that bacteria and metabolites differed significantly between the durable clinical benefit (DCB) and non-durable clinical benefit (NDB) groups, whereas the differences were smaller for fungi. The overall diversity of bacteria and fungi before treatment was higher in the DCB group than in the NDB group, and the difference in diversity began to change with the use of immunotherapy after 6-8 weeks. We also explored the alterations of gut microbes in the DCB and NDB groups, established 18 bacterial species models as predictive biomarkers for predicting whether immunotherapy is of sustained benefit (area under the curve=75.63%), and screened two species of bacteria (, and ) and one metabolite (galanthaminone) as prognostic biomarkers for predicting survival in patients with HCC treated with ICI. CONCLUSIONS: In this study, the status and characterization of the multi-kingdom microbiota, including gut bacteria, fungi, and their metabolites, were described by multiomics sequencing for the first time in patients with HCC treated with ICI. Our findings demonstrate the potential of bacterial taxa as predictive biomarkers of ICI clinical efficacy, and bacteria and their metabolites as prognostic biomarkers.