Nonstructural protein 3 (NS3) of hepatitis C virus (HCV) is a trypsin-like protease and is essential for processing of viral polyprotein. Accordingly, it is a potential target for anti-HCV drugs. Recently we could isolate RNA aptamers (G9-I, II, and III) which bind and inhibit NS3 protease using in vitro selection strategy. In addition, G9-I aptamer showed noncompetitive inhibition. In order to elucidate the binding site of G9-I aptamer in NS3 protease domain (delta NS3), we carried out alanine scanning mutagenesis at positive charged residues on the surface of delta NS3. The result of binding analysis by surface plasmon resonance measurements and protease inhibition assay clarified that Arg161 as well as Arg130 of delta NS3 are essential for interaction with G9-I aptamer. This region appears to be a potential targeting site for anti-HCV drugs.