Myxoma virus (MYXV), a Leporipoxvirus, is being developed as an oncolytic agent against various types of human cancers. It successfully infects and has oncolytic effects on cancer cells while remaining nonpathogenic to normal human cells and all other non-leporid species. To develop virus constructs and maximize their effectiveness against cancer cells, the interaction between virus and host should be well characterized. DEAD-box RNA helicase DDX3 was previously identified as an intrinsic host factor that regulates MYXV replication in human cancer cell lines. Here, it is reported that transient knockdown of DDX3 in human cancer cells significantly enhances MYXV replication and progeny virus production. In uninfected cells, DDX3 localizes throughout the cytoplasm of human cells; however, in the MYXV-infected cells, DDX3 localizes to the perinuclear region of the cells and forms granule-like particles. It is further demonstrated that DDX3 is likely enhancing the type-1 interferon (IFN) production as the expression of the cytokine is decreased when DDX3 is knocked down during MYXV virus infection. Thus, the absence of DDX3 significantly enhanced myxoma virus spread by reducing the production of type-1 IFN and IFN-mediated signaling. These results suggest that DDX3 is a potential cellular target for enhancing the oncolytic activity of MYXV in human cancers.