Conserved tryptophan-187 facilitates homodimerization of the influenza A virus NS1 effector domain. We generated a mutant influenza virus expressing NS1-W187R to destabilize this self-interaction. NS1-W187R protein exhibited lower dsRNA-binding activity, showed a temporal redistribution during infection, and was minimally compromised for interferon-antagonism. The mutant virus replicated similarly to wild-type in vitro, but was slightly attenuated for replication in mice, causing notably reduced morbidity/mortality. These data suggest biological relevance for the W187-mediated homotypic interaction of NS1.