There is increasing evidence that 2009 pandemic H1N1 influenza viruses have evolved after pandemic onset giving rise to severe epidemics in subsequent waves. However, it still remains unclear which viral determinants might have contributed to disease severity after pandemic initiation. Here, we show that distinct mutations in the 2009 pandemic H1N1 virus genome have occurred with increased frequency after pandemic declaration. Among those, a mutation in the viral hemagglutinin was identified that increases 2009 pandemic H1N1 virus binding to human-like α2,6-linked sialic acids. Moreover, these mutations conferred increased viral replication in the respiratory tract and elevated respiratory droplet transmission between ferrets. Thus, our data show that 2009 H1N1 influenza viruses have evolved after pandemic onset giving rise to novel virus variants that enhance viral replicative fitness and respiratory droplet transmission in a mammalian animal model. These findings might help to improve surveillance efforts to assess the pandemic risk by emerging influenza viruses.