Abstract: Type IV interferon (IFN-υ) is a recently discovered pivotal cytokine in the host defense against viral infections. Whether IFN-υ plays an important role and how is the mechanism in bacterial infections remain unknown. This study delves into the antibacterial and antiviral functions and mechanisms of grass carp (Ctenopharyngodon idella) IFN-υ (CiIFN-υ) in both in vivo and in vitro. Firstly, CiIFN-υ gene was identified and characterized in grass carp. Then, the immune expressions of CiIFN-υ exhibited a significant increase post bacterial challenge, which indicated CiIFN-υ responds to bacterial infections. Further, the eukaryotic recombinant expression plasmid of CiIFN-υ was constructed and transfected into FHM cells, supernatants were collected, incubated with four strains of bacteria respectively, spread plates and cultured for colony count, etc. The results indicated that CiIFN-υ demonstrates more potent antibacterial activity against Gram-negative bacteria versus Gram-positive bacteria, and aggregates Gram-negative bacteria but not Gram-positive bacteria. Furthermore, in vivo experiments verified the antibacterial function, with high survival rate, low tissue edema and damage, reduced tissue bacterial load and elevating proinflammatory response at early stage post bacterial infection. In addition, the antiviral function of CiIFN-υ were also confirmed in both in vitro and in vivo experiments, including crystal violet staining, survival rate, tissue viral burden, and qRT-PCR, etc. The present study highlights the antibacterial function and preliminary mechanism of IFN-υ, and the findings demonstrated that IFN-υ possesses dual functions against bacterial and viral infections.