Influenza A virus non-structural protein 1 (NS1) is the most important viral regulatory factor that controls cellular processes to facilitate viral replication. To gain further insight into the role of NS1, we tried to find novel cellular factors that interact with NS1. The complexes of NS1 and target proteins were pulled down from an infected cell lysate using anti-NS1 (A/Udorn/72) single-chain Fv and identified by peptide mass fingerprinting analysis. We identified nucleolin, a multifunctional major nucleolar protein, as a novel NS1-binding protein. The RNA-binding domain of NS1 was responsible for this binding, as judged by a GST (glutathione S-transferase) pull-down assay with the GST-fused functional domains of NS1. By laser confocal microscopy, we observed the co-localization of NS1 with nucleolin most clearly in the nucleoli, indicating that NS1 is interacting with nucleolin during infection. Our results suggest a novel function of NS1, namely, affecting cellular events via interaction with nucleolin.