Abstract: Type IV interferon (IFN-υ) is a recently discovered cytokine crucial for host defense against viral infections. However, the role and mechanisms of IFN-υ in bacterial infections remain unexplored. This study investigated the antibacterial and antiviral functions and mechanisms of grass carp (Ctenopharyngodon idella) IFN-υ (CiIFN-υ) both in vivo and in vitro. The CiIFN-υ gene was first identified and characterized in grass carp. Subsequently, the immune expression of CiIFN-υ significantly increased following bacterial challenge, indicating its response to bacterial infections. The eukaryotic recombinant expression plasmid of CiIFN-υ was then constructed and transfected into fathead minnow (FHM) cells. Supernatants were collected and incubated with four bacterial strains, followed by plate spreading and colony counting. Results indicated that CiIFN-υ exhibited more potent antibacterial activity against gram-negative bacteria compared to gram-positive bacteria and aggregated gram-negative bacteria but not gram-positive bacteria. In vivo experiments further confirmed the antibacterial function, showing high survival rates, low tissue edema and damage, reduced tissue bacterial load, and elevated proinflammatory response at the early stages of bacterial infection. In addition, the antiviral function of CiIFN-υ was confirmed through in vitro and in vivo experiments, including crystal violet staining, survival rates, tissue viral burden, and RT-qPCR. This study highlights the antibacterial function and preliminary mechanism of IFN-υ, demonstrating that IFN-υ possesses dual functions against bacterial and viral infections.