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The RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is the essential catalytic enzyme for viral genome replication. It initiates minus-strand RNA synthesis from a highly conserved 98-nt sequence, called the X-RNA, at the 3'-end of the plus-strand viral genome. In this study, we evaluated the antiviral effects of peptide nucleic acids (PNAs) targeting the X-RNA. Our in vitro RdRp assay results showed that PNAs targeting the three major stem-loop (SL) domains of X-RNA can inhibit RNA synthesis initiation. Delivery of X-RNA-targeted PNAs by fusing the PNAs to cell-penetrating peptides (CPPs) into HCV-replicating cells effectively suppressed HCV replication. Electrophoretic mobility shift assays revealed that the PNA targeting the SL3 region at the 5'-end of X-RNA dissociated the viral RdRp from the X-RNA. Furthermore, delivery of the SL3-targeted PNA into HCV-infected cells resulted in the suppression of HCV RNA replication without activation of interferon β expression. Collectively, our results indicate that the HCV X-RNA can be effectively targeted by CPP-fused PNAs to block RNA-protein and/or RNA-RNA interactions essential for viral RNA replication and identify X-RNA SL3 as an RdRp binding site crucial for HCV replication. In addition, the ability to inhibit RNA synthesis initiation by targeting HCV X-RNA using antisense PNAs suggests their promising therapeutic potential against HCV infection.