Recently identified hepatitis C virus (HCV) isolates that are infectious in cell culture provide a genetic system to evaluate the significance of virus-host interactions for HCV replication. We have completed a systematic RNAi screen wherein siRNAs were designed that target 62 host genes encoding proteins that physically interact with HCV RNA or proteins or belong to cellular pathways thought to modulate HCV infection. This includes 10 host proteins that we identify in this study to bind HCV NS5A. siRNAs that target 26 of these host genes alter infectious HCV production >3-fold. Included in this set of 26 were siRNAs that target Dicer, a principal component of the RNAi silencing pathway. Contrary to the hypothesis that RNAi is an antiviral pathway in mammals, as has been reported for subgenomic HCV replicons, siRNAs that target Dicer inhibited HCV replication. Furthermore, siRNAs that target several other components of the RNAi pathway also inhibit HCV replication. MicroRNA profiling of human liver, human hepatoma Huh-7.5 cells, and Huh-7.5 cells that harbor replicating HCV demonstrated that miR-122 is the predominant microRNA in each environment. miR-122 has been previously implicated in positively regulating the replication of HCV genotype 1 replicons. We find that 2'-O-methyl antisense oligonucleotide depletion of miR-122 also inhibits HCV genotype 2a replication and infectious virus production. Our data define 26 host genes that modulate HCV infection and indicate that the requirement for functional RNAi for HCV replication is dominant over any antiviral activity this pathway may exert against HCV.