This study investigated the function of the DEAD-box RNA helicase DDX3 in the life cycle of the hepatitis C virus (HCV) and in the cap- and HCV IRES-dependent translation. The following results were obtained: DDX3 supports HCV IRES-mediated translation but does not affect HCV RNA replication. DDX3 promotes overall translation. Experiments with reporter RNAs in Huh7 cells revealed that a depletion of DDX3 results in reduced IRES-dependent as well as cap-dependent translation. Immunoprecipitation experiments with purified components of the 43S PIC revealed eIF3 and the ribosomal 40S subunit as direct interaction partners of DDX3. To elucidate the precise function of DDX3 in the translation initiation process, translation-competent DDX3-depleted extracts were used. It has been demonstrated that DDX3 supports the assembly of 80S initiation complexes on a 5’cap mRNA and on an IRES-containing RNA, respectively. In contrast, the assembly of 48S initiation complexes was unaffected by DDX3.