The balance between cellular defense and viral escape determines the fate of influenza A virus (IAV) infection. Viral polymerase activity is critical for the replication and propagation of IAV. The antiviral strategies of host cells against IAV infection have not been fully elucidated. Here, we identified DCAF7 as an antiviral factor for IAV, which inhibits the replication of H1N1 and H3N2. Mechanistically, DCAF7 weakens the viral heterotrimer polymerase activity and restricts IAV replication and transcription. DCAF7 as a substrate recognition receptor forms a complete CRL4BDCAF7 E3 ligase with the CRL4B E3 complex to promote K48-linked polyubiquitination of the viral polymerase subunit PA at the K609 site and its degradation. We also showed that a specific cullin-RING E3 ligase (CRL) inhibitor MLN4924 upregulates the protein level of PA and promotes the replication of IAV in vivo. Moreover, activation of CUL4B by etoposide promotes the degradation of PA and inhibits IAV replication in vivo. Importantly, we found that viral NS1 protein decreases DCAF7 level to impair its antiviral efficacy. Taken together, these findings reveal a new mechanism of host resistance to IAV infection and suggest that regulation of the DCAF7-CRL4B axis is a potential target for antivirals.