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Despite the apparent lack of a feline influenza virus lineage, cats are susceptible to infection by influenza A viruses. Here, we characterized in vitro A/feline/Guangdong/1/2015, an H5N6 avian influenza virus recently isolated from cats. A/feline/Guangdong/1/2015 replicated to high titers and caused CPE in feline kidney cells. We determined that infection with A/feline/Guangdong/1/2015 did not activate the IFN-β promoter, but inhibited it by blocking the activation of NF-κB and IRF3. We also determined that the viral NS1 protein mediated the block, and that the dsRNA binding domain of NS1 was essential to perform this function. In contrast to treatment after infection, cells pretreated with IFN-β suppressed viral replication. Our findings provide an example of an H5N6 influenza virus suppressing IFN production, which might be associated with interspecies transmission of avian influenza viruses to cats.Our study demonstrates that the H5N6 virus isolated from cats can replicate in feline-origin cells.The NS1 protein of the H5N6 virus targets both NF-κB and IRF3 pathways to block the IFN mediated signaling pathway.Our findings also provide the first example of a novel H5N6 virus suppressing IFN production.Pretreated cells with IFN-β prevent viral replication in vitro and may provide a target for development of antiviral drugs.