Species differences in the host factor ANP32A/B result in the restriction of avian influenza virus polymerase (vPol) in mammalian cells. Efficient replication of avian influenza viruses in mammalian cells often requires adaptive mutations, such as PB2-E627K, to enable the virus to use mammalian ANP32A/B. However, the molecular basis for the productive replication of avian influenza viruses without prior adaptation in mammals remains poorly understood. We show that avian influenza virus NS2 protein help to overcome mammalian ANP32A/B-mediated restriction to avian vPol activity by promoting avian vRNP assembly and enhancing mammalian ANP32A/B-vRNP interactions. A conserved SUMO-interacting motif (SIM) in NS2 is required for its avian polymerase-enhancing properties. We also demonstrate that disrupting SIM integrity in NS2 impairs avian influenza virus replication and pathogenicity in mammalian hosts, but not in avian hosts. Our results identify NS2 as a cofactor in the adaptation process of avian influenza virus to mammals.