The nucleolus is a stress sensor associated with cell cycle progression and a central hub for the replication of pathogenic RNA viruses. However, the role of nucleolus in influenza A virus infection has not been well studied. Here we show that the interaction between NS1 protein of influenza A/Shantou/602/06 (H3N2) and nucleolin, a ubiquitous protein of nucleolus repressed RNA Pol I-dependent transcription via establishing hyper-methylation in the UCE of rRNA gene promoter. NS1 expressed cells showed significant association of ribosomal proteins with MDM2, and p53 accumulation, suggesting induced nucleolar stress. Disruption of the interaction of NS1 with nucleolin or overexpression of nucleolin in NS1 expressed cells revived RNA Pol I-dependent transcription, indicating nucleolin could be one target for NS1 to repress rRNA synthesis of host cells. Our present study suggests that NS1 protein of H3N2 could induce nucleolar stress based on epigenetic alteration of rRNA gene promoter via interaction with nucleolin.