DEAD-box helicase 5 (DDX5) plays a significant role in tumorigenesis and regulates viral replication of several viruses. An avian oncogenic herpesvirus, Marek’s disease virus (MDV), is widely known to cause immunosuppression and lymphoma in chickens. However, the underlying mechanisms of how DDX5 plays a role in viral replication remain unclear. In this study, we show that MDV inhibits the production of interferon beta (IFN-β) in chicken embryo fibroblasts (CEFs) by increasing the expression level and promoting the nuclear aggregation of DDX5. We further reveal how DDX5 down-regulates melanoma differentiation-associated gene 5/toll-like receptor 3 signaling through the fundamental transcription factor, interferon regulatory factor 1. MDV replication is suppressed, and the production of IFN-β is promoted in the DDX5 absented CEFs. Taken together, our investigations demonstrate that MDV inhibits IFN-β production by targeting DDX5-mediated signaling to facilitate viral replication, which offers a novel insight into the mechanism by which an avian oncogenic herpesvirus replicates in chicken cells.