Interactions between the viral surface glycoprotein hemagglutinin (HA) and the corresponding receptors on host cells is one important aspect of influenza virus infection. Mutations in the HA have been described to affect pathogenicity, antigenicity and the transmission of influenza viruses. Here, we detected polymorphisms present in the HA genes of two 2009 pandemic H1N1 isolates, A/California/04/2009 (Ca/09) and A/Mexico/4108/2009 (Mx/09), that resulted in amino acid changes at position 186 (S to P) and 194 (L to I) of the mature HA1 protein. Although not reported in the published 2009 pandemic H1N1 (H1N1pdm09) consensus sequence, the P186 genotype was more readily detected in primary infected and contact na?ve pigs when inoculated with a heterogeneous mixed stock of Ca/09. By reverse genetics we engineered Ca/09 and Mx/09 genomes by introducing the Ca/09 HA with two naturally occurring variants expressing S186/I194 (HA-S/I) and P186/L194 (HA-P/L), respectively. The Ca/09 HA with the combination of P186/L194 with either Ca/09 or Mx/09 backbones resulted in higher and prolonged viral shedding in na?ve pigs. This efficiency appeared to be more likely through an advantage in cell surface attachment rather than replication efficiency. Although these mutations occur within the receptor binding pocket and the Sb antigenic site, they did not affect serologic cross-reactivity. Relative increases of P186 in publically available sequences from swine H1N1pdm09 viruses supports the experimental data indicating this amino acid substitution confers an advantage in swine.