The hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) in its 5' untranslated region, the structure of which is essential for viral protein translation. The IRES includes a predicted pseudoknot interaction near the AUG start codon, but the results of previous studies of its structure have been conflicting. Using mutational analysis coupled with activity and functional assays, we verified the importance of pseudoknot base pairings for IRES-mediated translation and, using 35 mutants, conducted a comprehensive study of the structural tolerance and functional contributions of the pseudoknot. Ribosomal toeprinting experiments show that the entirety of the pseudoknot element positions the initiation codon in the mRNA binding cleft of the 40S ribosomal subunit. Optimal spacing between the pseudoknot and the start site AUG resembles that between the Shine-Dalgarno sequence and the initiation codon in bacterial mRNAs. Finally, we validated the HCV IRES pseudoknot as a potential drug target using antisense 2'-OMe oligonucleotides.