The neuraminidase (NA) inhibitor oseltamivir is widely used to treat patients infected with influenza viruses. An Ile-to-Val change at position 117 in H5N1 NA (NA-I117V) confers a reduction in susceptibility to oseltamivir carboxylate. However in vivo relevance and molecular basis of the decreased sensitivity mediated by this mutation are poorly understood.Methods.?We created single point mutant viruses with three genetically different backgrounds (i.e., one belonging to clade 1 and two belonging to clade 2.3.4) and evaluated the effects of the I117V mutation on oseltamivir susceptibility in vitro, in vivo, and in silico.Results.?The NA-I117V mutation conferred a slight reduction in susceptibility to oseltamivir in vitro (1.3- to 6.3-fold changes), although it did not substantially compromise NA enzymatic activity. Mice infected with I117V virus exhibited reduced susceptibility to oseltamivir and decreased survival in two of three virus pairs tested. Molecular dynamics simulations revealed that I117V caused the loss of hydrogen bonds between an arginine at position 118 and the carboxyl group of oseltamivir, leading to a lower binding affinity for oseltamivir.Conclusions.?Our findings provide new insight into the mechanism of NA-I117V mediated oseltamivir resistance in highly pathogenic H5N1 avian influenza viruses.