Phosphorylation of viral protein plays important roles in the influenza A virus (IAV) life cycle. By using mass spectrometry, we have identified tyrosine (Y) 132 as a phosphorylation site of matrix protein (M1) of influenza A/WSN/1933(H1N1). Phosphorylation at this site is essential to the process of virus replication by controlling the nuclear import of M1. We further demonstrate that the phosphorylated tyrosine is crucial for the binding of M1 to the nuclear import factor importin-α1, since any substitutions at this site severely reduce this protein-protein interaction and damage the importin-α1-mediated nuclear import of M1. Additionally, the tyrosine phosphorylation which leads to the nuclear import of M1 is blocked by the Janus kinase inhibitor. The present study reveals a pivotal role of this tyrosine phosphorylation in the intracellular transportation of M1, which controls the process of viral replication