One of the host-range determinants that favor the multiplication of influenza viruses in domestic poultry is the length of the neuraminidase (NA) stalk: a deletion of about 20 amino acids in the NA-stalk is frequently detected upon transmission of influenza A viruses from waterfowl to domestic poultry and was recently associated with increased virulence in chicken. Whether this modification has any effect in ducks is currently unknown. Using reverse genetics, a recombinant virus derived from a turkey H7N1 influenza virus isolate with a short-stalk neuraminidase, A/turkey/Italy/977/1999, and an NA stalk insertion variant (insNA) were produced. Four-week-old chickens that were inoculated via the intratracheal and oral routes with the insNA virus showed a better survival and a lower oro-pharyngeal excretion than those inoculated with the wt-virus. The two viruses did not differ as regards viral loads in the lungs, kidneys and caeca, but the short-stalk NA virus induced more necrotic lesions in the bronchial epithelium. Conversely, the insNA virus showed a higher cloacal excretion when inoculated to 4-wk-old ducks. Chickens inoculated with the less pathogenic, long-stalk NA-virus showed, at 2 d.p.i. in the lungs, significantly increased levels of mRNAs encoding IFNγ, IL-8, IL-15, IL-6, and to a lesser extent CCL5, when compared with their wt virus-inoculated counterparts. Both viruses infected efficiently a lung epithelial chicken cell line, in which they induced similar levels of transcription of the IFN-α and -β, Mx, and IL-8 genes. At the molecular level, the neuraminidase activity, which was null for the short-stalk NA virus in the chicken erythrocytes elution assay, was fully restored in the long-stalk NA virus. Overall, our data confirm that a shortened NA stalk is a strong determinant of adaptation and virulence of influenza viruses in chickens, and suggest reciprocally that a virus with a short-stalk NA is less fit for replication and shedding in ducks.