H5N1 avian influenza viruses demonstrate different phenotypes, such as pathogenicity after one or serial passage(s) in mammalian hosts or cells. To establish the molecular basis of these phenotypes, we cloned the isolates from the lung of mice infected with human A/Vietnam/1194/2004 (H5N1) influenza virus. Large-plaque isolates were less pathogenic to mice than small-plaque isolates. Genome sequencing revealed that the small-plaque and the large-plaque isolates were different in several amino acids. In order to assess their effects on pathogenicity in mice, two common amino acid changes in attenuated isolates, one in PB2 (I63T) and the other in PB1 (T677M) were inserted into the wild-type reverse genetic virus construct. The PB2 (I63T) or PB1 (T677M) mutation did not alone alter the phenotype of H5N1 virus, whereas the reverse genetic (rg) virus with both mutations was less pathogenic than the wild-type virus. Furthermore, the PB1 (T677M) mutation showed less replication efficiency though it had a higher polymerase activity. And the rg virus with PB2 (63T) mutation replicated as much as the wild-type virus. These results suggested that the C terminal of PB1 of H5N1 influenza virus mediated the virulence attenuation of H5N1 influenza virus in mice, associating with the N terminal of PB2. However, the role of the N terminal of PB2 in the virulence attenuation in mice remains unclear.