In the past, avian influenza viruses have crossed species’ barriers to trigger human pandemics by reassorting with mammal-infective viruses in intermediate livestock hosts. H5N1 viruses are able to infect pigs, and some of them have affinity for the mammalian type α-2,6-linked sialic acid airway receptor. By using reverse genetics, we systemically created 127 reassortant viruses between a duck isolate of H5N1, specifically retaining its hemagglutinin (HA) gene throughout, and a highly transmissible, human-infective H1N1 virus. We tested the virulence of the reassortants in mice as a correlate for virulence in humans, and tested transmissibility in guinea pigs, which have both avian and mammalian types of airway receptor. Transmission study showed that both polymerase PA gene and nonstructural protein NS gene of H1N1 virus made the H5N1 virus transmissible by respiratory droplet between guinea pigs, without death. Further experiments implicated other H1N1 genes in the enhancement of mammal-to-mammal transmission, including nucleoprotein (NP), neuraminidase (NA), and matrix (M), as well as mutations in H5 HA that improve affinity for human-like airway receptors. Hence, avian H5N1 subtype viruses do have the potential to acquire mammalian transmissibility by reassortment in current agricultural scenarios.