The emergence of influenza virus A pandemic H1N1 in April 2009 marked the first pandemic of the 21st century. In this study, we observed significant differences in the polymerase activities of two clinical 2009 H1N1 influenza A virus isolates from Chinese and Japanese patients. Sequence comparison of the three main protein subunits (PB2, PB1, and PA) of the viral RNA-dependent RNA polymerase complex and subsequent mutational analysis revealed that a single amino acid substitution (E206K) was responsible for the observed impaired replication phenotype. Further in vitro experiments showed that presence of PAE206K decreased the replication of influenza A/WSN/33 virus in mammalian cells and a reduction in the virus´s pathogenicity in vivo. Mechanistic studies revealed that PAE206K is a temperature-sensitive mutant associated with the inability to transport PB1-PA complex to the nucleus at high temperatures (39.5 °C). Hence, this naturally occurring variant in the PA protein represents an ideal candidate mutation for the development of live attenuated influenza vaccines.