Host genes involved in lipid metabolism are differentially affected during the early stages of hepatitis C virus (HCV) infection. Here we demonstrate that artificial up-regulation of fatty acid biosynthesis has a positive effect on the replication of the HCV full-length replicon when cells were treated with nystatin. Conversely, the HCV RNA replication was decreased when fatty acid biosynthesis was inhibited with 25-hydroxycholesterol and PDMP(D-threo-1-phenyl-2-decanoylamino-3- morpholino-1-propanol). In agreement with these results, the expression level of GlcT-1(ceramide glucosyltransferase), a host glucosyltransferase in the first step of GSL (glycosphingolipid) biosynthesis, was found to be closely associated with the expression and replication of HCV RNA. On the other hand, the viral RNA can also activate GlcT-1 in the early stage of viral RNA transfection in vitro. To identify viral factors that are responsible for GlcT-1 activation, we constructed ten stable Vero cell lines that express individual HCV proteins. Based on the analyses of these cell lines and transient transfection assay of the GlcT-1 promoter regions, we conclude that HCV proteins, especially NS5A and NS5B, have positive effects on the expression of GlcT-1. It is possible that NS5A and NS5B stimulate transcription factor(s) to activate the expression of GlcT-1 by increasing its transcription level.