To develop a novel attenuation strategy applicable to all influenza A viruses, we targeted the highly conserved protein-protein interaction of the viral polymerase subunits PA and PB1. We postulated that impaired binding between PA and PB1 would negatively affect trimeric polymerase complex formation, leading to reduced viral replication efficiency in vivo. As proof of concept, we introduced single or multiple amino acid substitutions into the protein-protein-binding domains of either PB1 or PA, or both, to decrease binding affinity and polymerase activity substantially. As expected, upon generation of recombinant influenza A viruses (SC35M strain) containing these mutations, many pseudo-revertants appeared that partially restored PA-PB1 binding and polymerase activity. These polymerase assembly mutants displayed drastic attenuation in cell culture and mice. The attenuation of the polymerase assembly mutants was maintained in IFNα/β receptor knock-out mice. As exemplified using a H5N1 polymerase assembly mutant, this attenuation strategy can be also applied to other highly pathogenic influenza A virus strains. Thus, we provide proof of principle that targeted mutation of the highly conserved interaction domains of PA and PB1 represents a novel strategy to attenuate influenza A viruses.