Several mechanisms underlying intertypic interference between co-infecting influenza types A and B viruses (IAV and IBV) have been proposed. We have recently described one in which IBV´s nucleoprotein (BNP) sequestered IAV´s nucleoprotein (ANP) and suppressed IAV polymerase and growth. However, its anti-IAV capacity and limitations have not been fully explored. Here, we showed that BNP´s inhibitory effect was more potent toward a wide array of avian IAVs, whereas human IAVs revealed moderate resistance. BNP sensitivity was largely determined by ANP´s residue 343 at the NP oligomerization interface. An avian IAV polymerase carrying an NP-V343L mutation switched from being highly BNP-sensitive to moderately BNP-resistant, and vice versa for a human IAV polymerase carrying a reverse mutation. To highlight its capacity, we demonstrated that the polymerases of highly-pathogenic H5N1 and the pandemic 2009 (H1N1) strains are strongly inhibited by BNP. Our work provides insights into lineage-specific sensitivity to BNP-mediated intertypic interference.