Enhanced translation of giardiavirus-luciferase chimeric mRNA in Giardia lamblia requires the initial 264-nt viral capsid coding region as a putative internal ribosomal entry site (IRES). Essential structural elements in this site include (1) a downstream box (DB) complementary to the anti-DB at the 3′ end of 16S-like rRNA, (2) stem-loops I, II, III, and IVA, and (3) a pentanucleotide 5′-UCUCC-3′ immediately downstream from stem loop IVA. A search for the structural role of the pentanucleotide suggested that it may form a pseudoknot with another pentanucleotide 5′-GGAGA-3′ in loop II. Alteration of the two pentanucleotides by site-directed mutagenesis resulted in a drastic reduction in translation of the transcript. But the loss was recovered by compensatory changes in the two sequences, suggesting Watson–Crick base pairings between them. Results from in vitro enzymatic and chemical structural probing supported the presence of such a pseudoknot 143 nt downstream from the initiation codon. Minor repositioning of this codon led invariably to a complete loss of translation, suggesting that the initiation site is confined within a rigid position defined by all the structural elements in the IRES including the pseudoknot. This is the first pseudoknot of its kind shown to play an important role in a downstream IRES of a viral transcript. The finding is particularly interesting because it could reflect a unique feature of translation initiation in Giardia, which is known to have exceedingly short (1–6 nt) 5′ untranslated regions in its mRNAs.