The 5' untranslated region (UTR) of hepatitis C virus (HCV), which is composed of four domains (I, II, III and IV) and a pseudoknot, is essential for translation and viral replication. Equine non-primate hepacivirus (EHcV) harbors a 5' UTR consisting of a large 5' -terminal domain (I), three additional domains (I', II and III), which are homologous to domains I, II and III, respectively, of HCV, and a pseudoknot, in the order listed. In this study, we investigated the roles of the EHcV 5' UTR in translation and viral replication. The internal ribosome entry site (IRES) activity of the EHcV 5' UTR was lower than that of the HCV 5' UTR in several cell lines due to structural differences in domain III. Domains I and III of EHcV were functional in the HCV 5' UTR in terms of IRES activity and the replication of the subgenomic replicon (SGR), although domain II was not exchangeable between EHcV and HCV for SGR replication. Furthermore, the region spanning domain I to I' of EHcV (5' proximal EHcV-specific region) improved RNA stability and provided HCV SGR with miR122-independent replication capability, while the EHcV domain I alone improved SGR replication and RNA stability irrespective of miR-122. These data suggest that the region spanning EHcV domain I to I' improves RNA stability and viral replication regardless of miR-122 expression. The 5' proximal EHcV-specific region may exert an inherent mechanism to facilitate viral replication in non-hepatic tissues.IMPORTANCE EHcV is the closest viral homolog to HCV among other hepaciviruses. HCV exhibits a narrow host range and a liver-specific tropism, while epidemiological reports suggest that EHcV infects the liver and respiratory organs of horses, donkeys and dogs. However, the mechanism explaining the differences in host or organ tropism between HCV and EHcV is unknown. In this study, our data suggest that the 5' untranslated region (UTR) of EHcV is composed of an internal ribosome entry site (IRES) element that is functionally exchangeable with HCV IRES elements. Furthermore, the 5' proximal EHcV-specific region enhances viral replication and RNA stability in an miR-122-independent manner. Our data suggest that the region upstream of domain II in the EHcV 5' UTR contributes to the differences in tissue tropism observed between these hepaciviruses.