Abstract Accumulating data suggest that tripartite motif-containing (TRIM) proteins participate in host responses to viral infections, either by acting as direct antiviral restriction factors or through regulating innate immune signaling of the host. Of >70 TRIMs, TRIM56 is a restriction factor of several positive-strand RNA viruses, including three members of the family Flaviviridae (yellow fever virus, dengue virus and bovine viral diarrhea virus) and a human coronavirus (OC43), and this ability invariably depends upon the E3 ligase activity of TRIM56. However, the impact of TRIM56 on negative-strand RNA viruses remains unclear. Herein, we show TRIM56 puts a check on replication of influenza A and B viruses in cell culture, but does not inhibit Sendai virus or human Metapneumovirus, two paramyxoviruses. Interestingly, the anti-influenza activity was independent of the E3 ligase activity, B-box, or coiled-coil domains. Rather, deletion of a 63-residue long, C-terminal tail portion of TRIM56 abrogated the antiviral function. Moreover, expression of this short C-terminal segment curtailed the replication of influenza viruses as effectively as that of full-length TRIM56. Mechanistically, TRIM56 was found to specifically impede intracellular influenza virus RNA synthesis. Consistent with this, a fraction of TRIM56 was re-distributed to the nucleus after IAV infection. Together, these data reveal novel antiviral activity of TRIM56 against influenza A and B viruses and provide novel insights into the mechanism by which TRIM56 restricts these medically important orthomyxoviruses. These findings raise the possibility that small molecules mimicking the C-terminal portion of TRIM56 could be developed into broad influenza antivirals.