Ubiquitination is a pivotal regulatory mechanism in host-virus interactions, playing essential roles in both antiviral defense and viral immune evasion, with E3 ubiquitin ligases serving as critical regulatory nodes. In this study, we systematically evaluated 26 candidate host E3 ubiquitin ligases for their involvement in influenza A virus (IAV) infection. Among these, potassium channel modulatory factor 1 (KCMF1) was identified as a novel negative regulator of IAV replication in vitro and in vivo. Mechanistically, KCMF1 interacts with the viral polymerase subunit PB1 and mediates its ubiquitination at lysine 653 (K653), triggering proteasomal degradation and consequent impairment of polymerase activity. A recombinant PR8 (H1N1) virus carrying a K653R substitution in PB1 exhibits enhanced replication and increased pathogenicity in both cells and mice. Furthermore, the inhibitory effect of KCMF1 on PR8 virus replication is dependent on the K653 residue of PB1. Importantly, viruses harboring the PB1 K653 mutation display marked resistance to favipiravir (T-705), an inhibitor of viral RNA-dependent RNA polymerase, suggesting that mutations at this site may influence antiviral drug sensitivity in circulating strains, and have potential implications for clinical treatment and viral surveillance. In conclusion, our findings identify KCMF1 as a host restriction factor that suppresses IAV replication via ubiquitination of PB1 at K653.