BACKGROUND: Healthy livers contain a large number of resident macrophages named Kupffer cells (KCs), which are partially replaced by infiltrating monocyte-derived macrophages (MoMFs) during acute or chronic liver injury. Despite extensive research, understanding macrophage heterogeneity, spatial distribution and interactions with other cells within the liver remains challenging. METHODS: This study employs sequential multiplex immunofluorescence staining, advanced image analysis and single-cell RNA sequencing (scRNA-seq) analysis to characterise macrophages in both healthy and diseased livers in mice. RESULTS: Our data revealed that liver KCs made up more than 80% of total immune cells in healthy mouse livers, while massive amounts of MoMFs infiltrated into the livers after acute and chronic liver injury. KCs were more abundant and larger in Zones 1 and 2 compared with Zone 3 in healthy livers. Zone 1 KCs exhibited higher phagocytic activity than Zone 2/3 KCs and MoMFs. We simultaneously evaluated cell proliferation and apoptosis on one slide and found that proliferation and apoptosis of KCs and MoMFs significantly increased in acutely injured livers. We also performed scRNA-seq to investigate liver macrophage gene expression in naïve and concanavalin A (ConA)-treated mice. MoMF clusters expanded following ConA treatment, while KCs remained stable. Macrophages were divided into distinct subtypes, including CONCLUSION: We developed a robust framework for studying liver macrophages