Given that co-infection of cells with equivalent titers of influenza A and B viruses (FluA and FluB) has been shown to result in suppression of FluA growth, it is possible that FluB-specific proteins might hinder FluA polymerase activity and replication. We addressed this possibility by individually determining the effect of each gene of FluB on the FluA polymerase assay and found that the nucleoprotein of FluB (NP(FluB)) inhibits polymerase activity of FluA in a dose-dependent manner. Mutational analyses of NP(FluB) suggest that functional NP(FluB) is necessary for this inhibition. Slower growth of FluA was also observed in MDCK cells stably expressing NP(FluB). Further analysis of NP(FluB) indicated that it does not affect nuclear import of NP(FluA). Taken together, these findings suggest a novel role of NP(FluB) in inhibiting replication of FluA, providing more insights into the mechanism of interference between FluA and FluB and the lack of reassortants between them.