Non-structural protein NS1 of influenza A viruses interacts with cellular factors through its N-terminal RNA-binding, middle effector and C-terminal non-structured domains. NS1 attenuates antiviral responses in infected cells and thereby secures efficient virus replication. Some influenza strains express C-terminally truncated NS1 proteins due to nonsense mutations in the NS1 gene. To understand the role of the NS1 C-terminal region in regulation of antiviral responses, we engineered influenza viruses expressing C-terminally truncated NS1 proteins using A/WSN/33(H1N1) reverse genetics and tested them in human macrophages and in mice. We showed that a WSN virus expressing NS1 with a 28 aa deletion from its C terminus is a more powerful inducer of antiviral responses than the virus expressing full-length NS1, or one with a 10 aa truncation of NS1 in vitro. Thus, our findings suggest that the C-terminal region of NS1 is essential for regulation of antiviral responses. Moreover, viruses expressing truncated NS1 proteins could be good vaccine candidates.