Some avian influenza (AI) viruses have a deletion of up to 20-30 amino acids in their NA stalk. This has been associated with changes in virus replication and host range. Currently prevalent H9N2 AI viruses only have a 2 or 3 amino acid deletion, which were detected in G1 and Y280 lineage viruses, respectively. The effect of an NA deletion on the H9N2 phenotype has not been fully elucidated. In this study, we isolated G1 mutants that carried an 8 amino acid deletion in their NA stalk. To systematically analyze the effect of NA stalk length and concomitant (de)glycosylation on G1 replication and host range, we generated G1 viruses with various NA stalk lengths and were either glycosylated or not glycosylated. The stalk length was correlated with NA sialidase activity, using low molecular weight substrates, and with virus elution efficacy from erythrocytes. G1 virus replication in avian cells and eggs was positively correlated with NA stalk length, but was negatively correlated in human cells and mice. NA stalk length modulated G1 virus entry in host cells, with shorter stalks enabling more efficient G1 entry into human cells. However, with an HA with higher α2,6 Sia affinity, the effect of NA stalk length on G1 virus infection was reversed, with shorter NA stalks reducing virus entry into human cells. These results indicated that that a balance between HA binding affinity and NA sialidase activity, modulated by NA stalk length, was required for optimal G1 virus entry into human airway cells.IMPORTANCE H9N2 avian influenza (AI) virus, one of the most prevalent AI viruses, has caused repeated poultry and human infections, posing a huge public health risk. The H9N2 virus has diversified into multiple lineages, with the G1 lineage most prevalent worldwide. In this study, we isolated G1 variants carrying an 8 amino acid deletion in their NA stalk, which was, to our knowledge, the longest deletion found in H9N2 viruses in the field. NA stalk length was found to modulate G1 virus entry into host cells, with the effects being species-specific and dependent on the corresponding HA binding affinity. Our results suggested that, in nature, H9N2 G1 viruses balance their HA and NA functions by the NA stalk length, leading to the possible association of host range and virulence in poultry and mammals during the evolution of G1 lineage viruses.