Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. Although several HCV protease/polymerase inhibitors were recently approved by U.S. FDA， the combination of antivirals targeting multiple processes of HCV lifecycle would optimize anti-HCV therapy and against potential drug-resistance. Viral entry is an essential target step for antiviral development， but FDA-approved HCV entry inhibitor remains exclusive. Here we identify serotonin 2A receptor (5-HTR) is a HCV entry factor amendable to therapeutic intervention by a chemical biology strategy. The silencing of 5-HTR and clinically available 5-HTR antagonist suppress cell culture-derived HCV (HCVcc) in different liver cells and primary human hepatocytes at late endocytosis process. The mechanism is related to regulate the correct plasma membrane localization of claudin 1 (CLDN1). Moreover， phenoxybenzamine (PBZ)， an FDA-approved 5-HTR antagonist， inhibits all major HCV genotypes in vitro and displays synergy in combination with clinical used anti-HCV drugs. The impact of PBZ on HCV genotype 2a is documented in immune-competent humanized transgenic mice. Our results not only expand the understanding of HCV entry， but also present a promising target for the invention of HCV entry inhibitor.