Pigs are susceptible to infection with both human and avian influenza A viruses and are considered intermediate hosts that facilitate virus reassortment. Although H5N1 virus has spread to a wide range of avian and mammalian species, data about swine H5N1 isolates are scarce. To determine whether Asian H5N1 influenza viruses had been transmitted to pigs, a total of 1,107 nasal swab samples from healthy swine were collected from 2008 to 2009 in Jiangsu province of eastern China. In this survey, two H5N1 viruses A/swine/Jiangsu/1/2008 (JS/08) and A/swine/Jiangsu/2/2009 (JS/09) were isolated and identified. Phylogenetic analysis showed that JS/08 and JS/09 belonged to clade 7 and clade 2.3.4, respectively, and shared over 99.0 % sequence identity with poultry H5N1 isolates of the same clade in China. Receptor specificity analysis also showed that both of the swine H5N1 isolates bound preferentially to avian-type receptors. However, experiments in mammals indicated that JS/09 was moderately pathogenic to mice without prior adaption, whereas JS/08 had limited ability to replicate. Our findings suggest that pigs are naturally infected with avian H5N1 virus and highlight the potential threat to public health due to adaption or reassortment of H5N1 virus in this species.