Hepatitis C virus is an enveloped, ssRNA virus, which infects 3% of the world population. No fully efficient therapy for treating hepatitis C exists. This is mainly due to the quasispecies structure of the RNA genome population, which favors the emergence of resistant viral variants. Despite the high variability rate, significant sequence and, more importantly, structure conservation can be found in the so-called functional genomic RNA domains, many of them with unknown roles for the consecution of the viral cycle. Such genomic domains are potential therapeutic targets. This study validates the use of RNA-based inhibitors (aptamers) as molecular tools to control the functionality of the cis-acting replication element (CRE) within the HCV genome. The CRE is an essential partner for viral replication. Also this structural domain is involved in the regulation of the protein synthesis. A set of forty-four RNA aptamers was assayed for the ability to interfere with the viral RNA synthesis in a subgenomic replicon system. Four aptamers emerged as potent inhibitors of HCV replication by direct interaction with specific and well-defined functional RNA domains of the CRE, yielding a decrease in the HCV genomic RNA levels higher than 90%. Concomitantly, one of them also promoted a significant increase in viral translation (>50%), likely by its interaction with the nucleotides surrounding the viral stop translation codon. The three remaining aptamers efficiently competed with the binding of the NS5B protein to the CRE, thus explaining their antiviral activity. Present findings confirm the potential of the CRE as an anti-HCV drugs target and support the use of aptamers as molecular tools for challenging the functionality of RNA domains in viral genomes.