PA-X is a fusion protein of influenza virus which plays a crucial role in modulating influenza virus-induced host innate immune response and subsequent pathogenicity. However, the potential mechanism of PA-X regulation of the host innate immune response remains largely unknown. It is well known that NF-κB signal pathway is crucial for the immediate early step of immune responses activation, while the specific role of PA-X in NF-κB transcriptional activity is totally unknown. In this study, we initially showed that PA-X inhibits NF-κB transcription that stimulated by poly(I:C). We then further determined that the inhibitory effect on NF-κB activation mediated by PA-X was characterized by restricting NF-κB p65 nuclear translocation and nuclear NF-κB p65 activity but not by impeding the phosphorylation of NF-κB p65. Correspondingly, PA-X decreases the amount of NF-κB signaling pathway-associated genes, including TNF-α, Nos2, IL-6 and IL-2. Moreover, PA-X also suppresses both the mRNA and protein expression level of IFN-β, suggesting the direct contribution of PA-X to the inhibition of NF-κB-regulated IFN-β expression. Together, our study sheds light on the potential molecular mechanisms underlying the regulation of host NF-κB activity by PA-X and also identifies a novel functional role for PA-X in counteracting the host innate immune response. However, further exploration of the more elaborate mechanism of PA-X-mediated inhibition of NF-κB activity and the associated signaling pathway may help to elucidate its precise mechanism of evading and subverting the host immune response.