Hepatitis C virus (HCV) is a leading cause of chronic liver disease. Identification of key host cellular factors involved in the HCV replication cycle is important for understanding disease pathogenesis and identifying novel antiviral therapeutic targets. The objective of this study was to identify novel host factors with important roles in the HCV replication cycle. Two approaches were used to select factors to test as potential HCV cofactors. The first approach investigated factors previously shown to facilitate the replication of other RNA viruses. This included Rab GTPases and the RNA binding proteins, Staufen, TIAL1 and TIA1. The second approach investigated factors observed to be upregulated during HCV infection following microarray expression profiling of HCV (JFH-1 clone) infected Huh7 hepatoma cells. Host genes controlling innate immunity, proliferation, lipid and protein metabolism and intracellular transport were observed to increase in expression, whilst genes controlling oxidative stress and cytoskeletal regulation decreased in expression. The expression patterns observed also indicated that HCV associated pathogenesis may be caused by HCV-induced gene expression changes. Further investigation into the upregulation of lipid synthesis genes observed during HCV infection demonstrated that intracellular lipid abundance also increased significantly, and may contribute to the development of hepatic steatosis in chronically infected patients. Host gene silencing by siRNA knockdown was used to investigate the potential cofactor role of the selected candidate host factors. Factors shown to be essential for effective HCV replication and secretion included those involved in lipid metabolism (TXNIP, CYP1A1 and CIDEC), intracellular transport (RAB2B, RAB4A, RAB11B, RAB27A/B, RAB33B and ABLIM3), and mRNA regulation (Staufen1). It was also shown that TXNIP may control lipid metabolite abundance during HCV replication, and Staufen1 may colocalise with HCV replication complexes to control HCV genome translation, replication or trafficking. Further investigation into the potential HCV cofactors identified should now be performed to improve understanding of the HCV replication cycle and enable identification of novel factors for antiviral targeting.