Neuraminidase inhibitors (NAIs) play a key role in the management of influenza. Given the limited number of FDA-approved anti-influenza drugs, evaluation of potential drug-resistant variants is of high priority. Two NA mutations, V116A and I117V, are found in ～0.6% of human, avian, and swine N1 isolates. Using the A/California/04/09-like (CA/04, H1N1) background, we examined the impact of V116A and I117V NA mutations on NAI susceptibility, substrate specificity, and replicative capacity in normal human bronchial (NHBE) cells and a human respiratory epithelial cell line (Calu-3). We compared the impact of V116A and I117V on the functional properties of NA and compared these mutations with that of previously reported NAI-resistant mutations, E119A, H275Y, and N295S. All NA mutations were genetically stable. None of the viruses carrying NA mutations grew to significantly lower titers than CA/04 in Calu-3?cells. In contrast, V116A, I117V, E119A, and N295S substitutions resulted in significantly lower viral titers (1.2 logs) than the parental CA/04 virus in NHBE cells. V116A conferred reduced sensitivity to oseltamivir and zanamivir (13.7-fold). When MUNANA, 3´SL, and 6´SL substrates were applied, we observed that V116A reduced binding ability for all substrates (13.9-fold) and I117V led to the significantly decreased affinity for MUNANA and 6´SL (4.2-fold). Neither mutation altered the catalytic efficiency (kcat/KM) in catalyzing 3´SL, but the efficiency in catalyzing MUNANA and 6´SL was significantly decreased: only ～34.7% compared to the wild-type NA. The efficiencies of NAs with E119A, H275Y, and N295S mutations to catalyze all substrates were ～19.4% of the CA/04 NA. Our study demonstrates the direct effect of drug-resistant mutations located inside or adjacent to the NA active site on NA substrate specificity.