Hepatitis C virus (HCV) is a major cause of global morbidity, causing long-term pathologies, including cirrhosis and hepatocellular carcinoma. While hepatocytes are the major site of viral replication, the liver contains multiple non-parenchymal cells that regulate the hepatic microenvironment and may affect HCV infection in vivo. Current understanding of the role of non-parenchymal cells in HCV infection is limited. Therefore, this project aimed to establish co-culture systems that allowed investigations into interactions between hepatocytes and non-parenchymal cells, and how these interactions affected HCV infection. The results showed that in co-culture, activated liver myofibroblasts (aLMFs) negatively regulate HCV entry, replication and spread of infection in a cell contact dependent manner. Soluble factors, including extracellular matrix proteins, and common antiviral pathways did not induce this effect. Instead, we found that aLMFmodulated cell-contact affected hepatocyte membrane receptor dynamics, reducing the mobility of the HCV receptor, CD81, impairing viral entry and replication. In addition, we found that aLMF surface expressed VAP-1 also significantly reduced virus infection independently of receptor modulation. These findings greatly improved our understanding of how the interactions between hepatic cells affect HCV, highlighting the importance of non-parenchymal cells in mediating infection in the liver microenvironment.