Influenza virus non-structural protein 1 (NS1) counteracts host antiviral innate immune responses by inhibiting Retinoic acid inducible gene-I (RIG-I) activation. However, whether NS1 also specifically regulates RIG-I transcription is unknown. Here, we identify a CCAAT/Enhancer Binding Protein beta (C/EBPβ) binding site in the RIG-I promoter as a repressor element, and show that NS1 promotes C/EBPβ phosphorylation and its recruitment to the RIG-I promoter as a C/EBPβ/NS1 complex. C/EBPβ overexpression and siRNA knockdown in human lung epithelial cells resulted in suppression and activation of RIG-I expression respectively, implying a negative regulatory role of C/EBPβ. Further, C/EBPβ phosphorylation, its interaction with NS1 and occupancy at the RIG-I promoter was associated with RIG-I transcriptional inhibition. These findings provide an important insight into the molecular mechanism by which influenza NS1 commandeers RIG-I transcriptional regulation and suppresses host antiviral responses.