Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a heterodimer and mediate receptor interactions and viral fusion. Both E1 and E2 are targets of the neutralizing antibody (NAb) response and are candidates for the production of vaccines that generate humoral immunity. Previous studies have demonstrated that the N-terminal hypervariable region 1 (HVR1) can modulate the neutralization potential of MAbs but no information is available for the influence of HVR2 or the intergenotypic variable region (igVR) on antigenicity. In this study, we examined how the variable regions influence the antigenicity of E2661 using a panel of monoclonal antibodies (MAbs) raised to E2661, E2661 lacking HVR1, HVR2 and the igVR (Delta123), and well-characterized MAbs isolated from infected humans. We show for a subset of both neutralizing and non-neutralizing MAbs, that all three variable regions decrease the ability of MAbs to bind E2661 and reduce the ability of MAbs to inhibit E2-CD81 interactions. In addition, we describe a new MAb towards the 411-428 region of E2 (MAb24) that demonstrates broad neutralization against all 7 genotypes of HCV. The ability of MAb24 to inhibit E2-CD81 interactions is strongly influenced by the three variable regions. Our data suggest that HVR1, HVR2 and the igVR modulate exposure of epitopes on the core domain of E2 and their ability to prevent E2-CD81 interactions. These studies suggest that the function of HVR2 and the igVR is to modulate antibody recognition of glycoprotein E2 and may contribute to immune evasion. IMPORTANCE: This study reveals conformational and antigenic differences between the Delta123 and intact E2661 glycoproteins and provides new structural and functional data about the three variable regions and their role in occluding neutralizing and non-neutralizing epitopes on the E2 core domain. The variable regions may therefore function to reduce the ability of HCV to elicit NAbs directed to the conserved core domain. Future studies aimed at generating a three dimensional structure for intact E2 containing HVR1 and the adjoining major neutralization epitope, and HVR2 will reveal how the variable regions modulate antigenic structure.