Exposure to poultry in live poultry markets is strongly associated with human infection with avian influenza virus. To effectively prevent the transmission of viruses from live poultry to humans, people have been forced to change their living habits from purchasing live poultry for consumption to purchasing freshly slaughtered poultry after the permanent closure of live poultry markets in China. In this study, we reported a case of human infection by the H5N6 virus in Hangzhou after exposure to a freshly slaughtered chicken, defying the traditional hypothesis that human infection requires a history of exposure to live poultry and indicating a novel route of infection. Rapid genomic characterization of H5N6 influenza A variants from the patient and the associated environment suggested that these viral variants were of avian origin, belonged to clade 2.3.4.4b H5 and were adapting to the human host after infection. Comparative analysis of the local H5N6 genomes showed that viral contamination in the associated environment and the poultry market was complex. Considering this case of H5N6 infection, conducting surveillance for any possible new avian influenza virus reassortment spillover to humans or other animal species is critical, and awareness of the risk of exposure to possible viral variants from infected slaughtered poultry or the associated environment must be seriously improved.Highlights We reported the first case of human infection with avian-origin influenza A (H5N6) virus in Zhejiang Province, southeastern China.Rapid genomic characterization of H5N6 influenza A variants from a patient and the associated environment suggested that these viral variants were of avian origin and were adapting to the human host after infection.Comparative analysis of the H5N6 genomes showed that viral contamination in the associated environment and poultry market was complex.Considering this case of H5N6 infection, the risk of exposure to possible viral variants from infected slaughtered poultry or the associated environment must be seriously considered.