Interferon regulatory factor-1 (IRF-1) mediates an antiviral state in cells by regulating the expression of the interferon (IFN-alpha/beta) system. To elucidate the role of IRF-1 in fish during virus infections, we constructed a recombinant plasmid of the Japanese flounder, Paralichthys olivaceus IRF-1 (JF IRF-1) under the control of the cytomegalovirus (CMV) immediate/early enhancer promoter. The antiviral mechanism of JF IRF-1 was studied using transfection experiments in a homologous cell line. Here, we show that cell supernatants obtained from transiently transfected cells enhanced cell viability of a heterologous cell line upon incubation, reduced the titers of hirame rhabdovirus (HIRRV) and viral hemorrhagic septicemia virus (VHSV), and possessed cytokine-like activity, as shown by their ability to protect cells against virus infections. The supernatants also inhibited the replication of the rhabdoviruses during the early stages of infection as indicated by the reduction of viral titers in the presence of the supernatants obtained from the transfected cells. Further analysis showed that the cell culture supernatants contain cytokine-like substances that possess acid-labile and temperature-resistant properties. These results indicate that JF IRF-1 induces an antiviral state in cells by mediating the production of cytokine-like substances. Thus, JF IRF-1 might be useful as an adjuvant in the development of DNA vaccines against commercially important viral pathogens in Japanese flounder aquaculture.