In Giardia, enhanced translation of luciferase mRNA, flanked between the 5’- untranslated region (UTR) and 3’-end of giardiavirus (GLV) transcript, requires presence of the initial 264 nucleotide (nt) viral capsid-coding region. By introducing the transcripts of dicistronic viral constructs into Giardia, we demonstrated that the 264 nt downstream region alone is insufficient to function as an IRES without including a portion of the 5’-UTR as well. Deletion analysis showed that efficient internal initiation requires the last 253 nts (nt 114-367) of 5’-UTR upstream of the initiation codon in combination with the downstream 264 nts. Specific mutations that deleted the predicted structural elements in either the 5’-UTR or the 264 nt capsid-coding region completely abolished the IRES-mediated translation of downstream cistron, suggesting that the IRES activity requires the presence of these structures in both regions. Mutations that abolished translation of the first cistron did not, however, affect the IRES-mediated translation of the second cistron, indicating that this IRES-mediated translation is independent of the translation of the upstream cistron. This is, to our knowledge, the first reported identification of a viral IRES with an estimated size of 549 nts that extends to both sides of the initiation site.