Influenza A virus causes significant morbidity in swine, resulting in a substantial economic burden. Swine influenza virus (SIV) infection also poses important human public health concerns. It has been shown that conversion of the haemagglutinin (HA) cleavage site from a trypsin-sensitive motif to an elastase-sensitive motif resulted in attenuated viruses in mouse models. However, application of this attenuation approach in a natural host has not been achieved yet. Here, we report that using reverse genetics, we generated two mutant SIVs derived from strain A/SW/SK/18789/02 (H1N1). Mutant A/SW/SK-R345V carries a mutation from arginine to valine at aa 345 of HA. Similarly, mutant A/SW/SK-R345A encodes alanine instead of arginine at aa 345 of HA. Our data showed that both mutants are solely dependent on neutrophil elastase cleavage in tissue culture. These tissue culture-grown mutant SIVs showed similar growth properties in terms of plaque size and growth kinetics to the wild-type virus. In addition, SIV mutants were able to maintain their genetic information after multiple passaging on MDCK cells. Furthermore, mutant SIVs were highly attenuated in pigs. Thus, these mutants may have the potential to serve as live attenuated vaccines.