Hepatitis C virus (HCV) is a major cause of chronic hepatitis and liver carcinoma and new therapies based on novel targets are needed. The tight junction protein Claudin 1 (CLDN-1) is essential for hepatitis C virus (HCV) cell entry and spread and anti-CLDN-1 rat and mouse monoclonal antibodies are safe and effective in preventing and treating HCV infection in a human liver chimeric mouse model. To accelerate translation of these observations into a novel approach to treat HCV infection and disease in humans, we screened a phage display library of human single chain antibody fragments (scFv) by using a panel of CLDN-1-positive and -negative cell lines and identified phage specifically binding to CLDN-1. Twelve clones, showing the highest levels of binding, were converted into human IgG4. Some of these mAbs display low nanomolar affinity, and inhibit infection of human hepatoma HuH7.5 cells by different HCV isolates in a dose-dependent manner. Cross-competition experiments identified six inhibitory mAbs that recognized distinct epitopes. Combination of the human anti-SRB1 mAb C-1671 with these anti-CLDN-1 mAbs could either increase or reduce inhibition of HCVcc infection in vitro. These novel human anti-CLDN-1 mAbs are potentially useful to develop a new strategy for anti-HCV therapy and lend support to the combined use of antibodies targeting the HCV receptors CLDN-1 and SRB1, but indicate that care must be taken in selecting the proper combination.