Pseudorabies virus (PRV) is a pathogen of swine resulting in devastating disease and economic losses worldwide. Resveratrol (Res) exhibits inhibitory activity against a wide range of viruses. Despite these important advances, the molecular mechanism(s) by which Res exerts its broad biological effects have not yet been elucidated. In this paper, the antiviral activity of Res against PRV and its mechanism of action were investigated. The results showed that Res potently inhibited PRV replication in a dose-dependent manner, with a 50% inhibition concentration of 17.17 μM. The inhibition of virus multiplication in the presence of Res was not attributed to direct inactivation or inhibition of viral entry into the host cells but to the inhibition of viral multiplication in host cells. Further studies demonstrated that Res is a potent inhibitor of both NF-κB activation and NF-κB-dependent gene expression through its ability to inhibit IκB kinase activity, which is the key regulator in NF-κB activation. Thus, the inhibitory effect of Res on PRV-induced cell death and gene expression may be due to its ability to inhibit the degradation of IκB kinase. These results provided a new alternative control measure for PRV infection and new insights into the antiviral mechanism of Res.