Neuraminidase (NA) mutations conferring resistance to NA inhibitors (NAIs) are expected to occur at framework or catalytic residues of the NA enzyme. Numerous clinical and in vitro reports already described NAI-resistant A(H1N1)pdm09 variants harboring various framework NA substitutions. By contrast, variants with NA catalytic changes remain poorly documented. Herein, we investigated the effect of R152K and R368K NA catalytic mutations on the NA enzyme properties, in vitro replicative capacity and virulence of A(H1N1)pdm09 recombinant viruses. In NA inhibition assays, the R152K and R368K substitutions resulted in reduced inhibition [10- to 100-fold increases in IC50 vs the wild-type (WT)] or highly reduced inhibition (>100-fold increases in IC50) to at least 3 approved NAIs (oseltamivir, zanamivir, peramivir and laninamivir). Such resistance phenotype correlated with a significant reduction of affinity observed for the mutants in enzyme kinetics experiments [increased Km from 20±1.77 for the WT to 200.8±10.54 and 565.2±135μM(P<0.01) for the R152K and R368K mutants, respectively]. The R152K and R368K variants grew at comparable or even higher titers than the WT in both MDCK and ST6GalI-MDCK cells. In experimentally-infected C57BL/6 mice, the recombinant WT and the R152K and R368K variants induced important signs of infection (weight loss) and resulted in mortality rates of 87.5%, 37.5% and 100%, respectively. The lung viral titers were comparable between the three infected groups. While the NA mutations were stable, an N154I substitution was detected in the HA2 protein of the R152K and R368K variants after in vitro passages as well as in lungs of infected mice. Due to the multi-drug resistance phenotypes and conserved fitness, the emergence of NA catalytic mutations accompanied with potential compensatory HA changes should be carefully monitored in A(H1N1)pdm09 viruses.