The entry pathway of the hepatitis C virus (HCV), a major human pathogen, into the cell is incompletely defined. To better characterize this viral life cycle stage, we screened a small interfering RNA library dedicated to the membrane trafficking and remodeling with the infection model of Huh-7.5.1 cells by HCV pseudoparticles (HCVpp). Results showed that the down-regulation of different factors implied in clathrin-mediated endocytosis (CME) inhibits HCVpp cell infection. In addition, knockdown of the phosphatidylinositol 4-kinase type III-alpha (PI4KIIIalpha) prevented infection by HCVpp or by cell-culture grown JFH-1-based HCV. Moreover, the replication activity of an HCV replicon was also affected by the PI4KIIIalpha knockdown. Additional investigations on the different members of the PI4K family revealed that the presence of PI4KIIIbeta in the host cells influenced their susceptibility to HCVpp infection and their capacity to sustain the HCV replication. The PI4KIII involvement during the HCV life cycle seemed to occur by other ways than the control of the CME or of the membranous expression of HCV receptors. Finally, our library screening completed data on the CME-dependant entry route of HCV and identified 2 kinases, PI4KIIIalpha and beta, as relevant potential therapeutic targets.