The technique of in vitro selection is a powerful tool for the isolation of nucleic-acid ligands (aptamers) that bind target molecules. RNA aptamers provide much information about the interaction between ligands and their targets, such as the relationship between sequence and structure. Furthermore, by inhibiting the function of a disease-related target molecule, aptamers can act as potential drug candidates. The hepatitis C virus (HCV) has a positive, single-stranded RNA genome, and translation starts within the internal ribosome entry site (IRES) in a cap-independent manner. The IRES is well conserved in HCV subtypes, and is a possible target site for anti-HCV drugs. We used an in vitro selection procedure to isolate RNA aptamers capable of binding to the IRES. We identified an aptamer consensus sequence that was complementary to the specific stem–loop structure of a highly ordered RNA and therefore likely to provide stable interaction with the target molecule. One of the aptamers bound to an important functional region of the IRES, and inhibited IRES-dependent translation both in vitro and in vivo. We propose that in vitro selection is a useful technique for drug design against target molecules.