Duck hepatitis virus (DHV-1) is a worldwide distributed picornavirus that causes acute and fatal disease in young ducklings. Recently, the complete genome of DHV-1 has been determined and comparative sequence analysis has shown that possesses the typical picornavirus organization but exhibits several unique features. For the first time, we provide evidence that the 626-nucleotide-long 5'-UTR of the DHV-1 genome contains an internal ribosome entry site (IRES) element that functions efficiently both in vitro and in mammalian cells. The prediction of the secondary structure of the DHV-1 IRES shows significant similarity to the hepatitis C virus (HCV) IRES. Moreover, similarly to HCV IRES, DHV-1 IRES can direct translation initiation in the absence of a functional eIF4F complex. We also demonstrate that the activity of the DHV-1 IRES is modulated by a viral coding sequence located downstream of the DHV-1 5'-UTR, which enhances DHV-1 IRES activity both in vitro and in vivo. Furthermore, mutational analysis of the predicted pseudo-knot structures at the 3'-end of the putative DHV-1 IRES supported the presence of conserved domains II and III and, as it has been previously described for other picornaviruses, these structures are essential for keeping the normal internal initiation of translation of DHV-1.