Pseudorabies virus (PRV) is a member of the genus Varicellovirus , family Herpesviridae and causes Aujeszky's disease to lead to huge economic losses in the global pig industry. The Non-POU domain-containing octamer-binding protein (NONO), as a Drosophila behavior/human splicing (DBHS) protein, plays a key role in multiple biological functions in cells, including transcriptional regulation, RNA splicing, DNA repair and so on. However, whether swine NONO (sNONO) inhibits PRV infection is less understood. In this study, we showed that sNONO was a crucial host factor for antagonizing PRV infection and positive regulated transcription levels of ISGs. After PRV infection, sNONO enhanced the activation of IFN-β promoter and IFN-β expression. Furthermore, knockout of sNONO in PAM-KNU cells impaired activation of type I IFN pathway and increased PRV propagation. Taken together, we have first elucidated the anti-PRV function and mechanism of sNONO, which may provide a new strategy for preventing DNA virus infection. • sNONO was a crucial host factor for antagonizing PRV infection and positive regulated transcription levels of ISGs. • sNONO enhanced the activation of IFN-β promoter and IFN-β expression after PRV infection. • Knockout of sNONO in PAM-KNU cells impaired activation of type I IFN pathway and increased PRV propagation. • The newly constructed sNONO KO-PAM-KNU cell line could be used to study biological function of sNONO and viral pathogenesis. [ABSTRACT FROM AUTHOR]