The species-specific acidic nuclear phosphoprotein 32?kDa members (ANP32A/B) play a key role in restricting the function of avian influenza virus polymerase in mammalian host. Avian influenza viruses, upon replicating in humans, may acquire critical amino acid substitutions in the basic polymerase 2 (PB2) protein (e.g. E627?K and D701N) that enhance the viral polymerase’s functional compatibility with human ANP32A and ANP32B proteins, thereby augmenting viral replication efficiency and increasing pathogenic potential in humans. However, certain H5N1 avian influenza viruses have demonstrated the capacity to establish productive human infections in the absence of the canonical PB2-627?K or PB2-701N adaptive mutations; the underlying molecular mechanism remains incompletely characterized. In this study, we found that the two H5N1 viruses, namely A/chicken/LN/SD035/2018 (LN35) and A/duck/JL/S1261/2019 (JL261), were genetically similar, but their pathogenicity for mice was different. By evaluating a series of single-gene reassortant viruses and mutants in mice, we confirmed that the PB2-384L/443R/460M characteristics are crucial for LN35 to maintain high lethality in mice. We further revealed that the polymerase of H5N1 avian virus bearing PB2-384L/443R/460M signature could efficiently utilize human ANP32A/B. Moreover, we found that this PB2-384L/443R/460M signature positively regulates H5N1 vRNP-human ANP32A/B interaction and vRNP assembly. Our findings indicate that key amino acid substitutions in the PB2 gene of the H5N1 virus may emerge in avian host and then augment its capacity to interact with human ANP32A/B protein, underscoring the substantial zoonotic and public health risks associated with continued circulation and evolution of H5N1 in avian populations.