Objective: Hepatocellular carcinoma (HCC) tumour microenvironment (TME) is highly complex with diverse cellular components organizing into various functional units (CN). And we wanted to define CN of HCC while preserving the TME architecture, based on which, potential targets for novel immunotherapy could be identified. Design: A highly multiplexed imaging mass cytometry (IMC) panel was designed to simultaneously quantify 36 biomarkers of tissues from 134 HCC patients and 7 healthy donors to generate 562 highly multiplexed histology images at single-cell resolution. Different function units were defined by topological analysis of TME. CN relevant to the patients’ prognosis was identified as specific target for HCC therapy. Transgenic mouse models were used to validate the novel immunotherapy target for HCC. Results: Three major types of intra-tumour areas with distinct distribution patterns of tumoral, stromal and immune cells were identified. 22 cellular metaclusters and 16 CN were defined. CN composed of various types of cells formed regional function units and the regional immunity was regulated reversely by resident Kupffer cells and infiltrating macrophages with pro- and anti-tumour function, respectively. Depletion of Kupffer cells in mouse liver largely enhances the T cell response, reduces liver tumour growth, and sensitizes the tumour response to anti-PD-1 treatment. Conclusion: Our findings reveal for the first time the various topological function units of HCC TME, which also presents the largest depository of pathological landscape for HCC. This work highlights the potential of Kupffer specific targeting rather than overall myeloid cell blocking as a novel immunotherapy for HCC treatment.