IRF9 is a key factor in the JAK-STAT pathway. Under the stimulation of type I IFN, IRF9 interacts with STAT1 and STAT2 to form the IFN-I-stimulated gene factor 3 (ISGF3) which activates the transcription of ISG. However, many studies also showed that the dimmer IRF9/STAT2 rather than the tripolymer IRF9/STAT1/STAT2 acts as the ISGF3 in cells in response to IFN signals. In the present study, the full-length cDNA sequence of IRF9 (termed CiIRF9, KT601055) and STAT2 (term CiSTAT2, KT781914) from grass carp were cloned and identified. A low level of constitutive expression of CiIRF9 was detected by RT-PCR in grass carp tissues, but it was significantly up-regulated by LPS and poly I:C stimulation. In vitro, a high-affinity interaction between CiIRF9 and the promoter of CiIFN or CiPKR was demonstrated by gel mobility shift assay. In vivo, the promoter activities of CiIFN and CiPKR were not only increased by transient transfection of CiIRF9, but also prominently increased by co-transfection of CiIRF9 and CiSTAT2. Moreover, the interaction of CiIRF9 and CiSTAT2 was further investigated by in vivo and in vitro protein interaction assays. Recombinant CiIRF9 and CiSTAT2, both tagged with FLAG (or HA), were expressed in HEK 293T cells by transient transfection experiment. Co-immunoprecipitation assays showed that CiIRF9 can interact with CiSTAT2 in vivo. Soluble GST-ST2-936 (containing the N-terminal and coiled-coil domain of CiSTAT2) was expressed and purified from E. coli. A GST pull-down assay suggested that GST-tagged ST2-936 efficiently bound to FLAG-tagged IRF9. The data indicated that interaction of IRF9 and STAT2 synergistically up-regulated the transcriptional level of IFN and ISG genes.