Influenza A virus is an important pathogenic virus known to induce host cell cycle arrest in G0/G1 phase and create beneficial conditions for viral replication. However, how the virus achieves arrest remains unclear. We investigated the mechanisms underlying this process and found that the non-structural protein 1 (NS1) is required. Based on this finding, we generated a viable influenza A virus (H1N1) lacking the entire NS1 gene to study the function of this protein in cell cycle regulation. Besides some cell cycle regulators were changed, the concentration and activity of RhoA protein, which is thought to be pivotal for G1/S phase transition, was also decreased with over-expressing NS1. At the same time, cell cycle regulator pRb, the downstream of RhoA kinase´s phosphorylation level was decreased in an NS1-dependent manner. These findings indicate that the NS1 protein induces G0/G1 cell cycle arrest mainly through interfering with the RhoA/pRb signalling cascade, thus providing favorable conditions for viral protein accumulation and replication. We further investigated the NS1 protein of avian influenza virus (H5N1) and found it also can decrease the expression and activity of RhoA, suggesting that the H5N1 virus may affect the cell cycle through the same mechanism. The NS1/RhoA/pRb cascade which can induce the G0/G1 cell cycle arrest identified here provides a unified explanation for the seemingly different NS1 functions involved in viral replication events. Our findings shed light on the mechanism of influenza virus replication and open new avenues for understanding the interaction between pathogens and hosts.