Since the emergence of the 2009 pandemic (H1N1) virus (2009/H1N1) in April 2009, cases of transmission from humans to pigs have been reported frequently. In our previous studies, four 2009/H1N1 variants were isolated from pigs. To better understand the phenotypic differences of the pig isolates compared with the human isolate, in this study mice were inoculated intranasally with different 2009/H1N1 viruses, and monitored for morbidity, mortality, and viral replication, cytokine production and pathological changes in the lungs. The results show that all isolates show effective replication in lungs, but varying in their ability to cause morbidity. In particular, the strains of A/swine/Nanchang/3/2010 (H1N1) and A/swine/Nanchang/F9/2010 (H1N1) show the greatest virulence with a persisting replication in lungs and high lethality for mice, compared with the human isolate A/Liaoning /14/2009 (H1N1), which shows low virulence in mice. Furthermore, the lethal strains could induce more severe lung pathological changes and higher production of cytokines than that of other strains at an early stage. Amino acid sequence analysis illustrates prominent differences in viral surface glycoproteins and polymerase subunits between pig isolates and human strains that might correlate with their phenotypic differences. These studies demonstrate that the 2009/H1N1 pig isolates exhibit heterogeneous infectivity and pathogencity in mice, and some strains possess an enhanced pathogenicity compared with the human isolate.