Background Hepatitis C virus (HCV) remains a major global health challenge, largely due to the absence of robust animal models that recapitulate this disease, hindering mechanistic studies and vaccine development. This study employed tree shrews to determine the molecular profiles of distinct liver cell populations following HCV infection. Methods Tree shrews received intrahepatic injections of the HCV JFH1 strain and were analysed at two timepoints: 1 week post-inoculation (acute) and 111 weeks post-inoculation (long-term). Viral load was assessed up to 30 weeks. Liver pathology was assessed via haematoxylin and eosin (H&E) staining. Single-cell transcriptomic profiling was performed on liver tissues, while tree shrew primary hepatocytes and ITH6 hepatic cells, as well as human Huh7 cells, were used to characterise cellular changes following HCV infection. Findings Sixteen major cell types were identified in 157,298 liver cells across HCV-inoculated and control tree shrews. Acute inoculation triggered widespread induction of interferon-stimulated genes (ISGs) across all cell types, including the periportal ISG high hepatocyte subcluster, and concurrent reduction in metabolic gene expression in hepatocytes in vivo. Impaired glucose metabolism, as confirmed in HCV-exposed ITH6 cells, likely contributed to this metabolic shift. Erythroblasts were exclusively detected during acute inoculation and exhibited enhanced intercellular communication signatures. Among neutrophils, three distinct subclusters were identified, one of which displayed elevated expression of neutrophil extracellular trap (NET) markers and enhanced NET formation in inoculated livers. Interpretation This study provides a comprehensive single-cell transcriptomic landscape of HCV-inoculated tree shrew livers. These findings underscore the use of the tree shrew model for advancing mechanistic understanding of HCV pathogenesis, as well as its relevance for therapeutic and vaccine development. Copyright (c) 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).