Since the reemergence of highly pathogenic avian influenza virus H5N1, it caused disease in 20 people with 13 deaths in mainland of China. On February 21, 2006, the first suspected human case in Zhejiang province was reported. Pathogenic analyses, including reverse transcriptase polymerase chain reaction (RT-PCR), real-time RT-PCR, and virus isolation, were carried out to confirm the pathogen from tracheal aspirate specimen. In addition, antibody in serum sample was detected using hemagglutination-inhibition (HI). Results revealed that nucleic acid extracted from the tracheal aspirate specimen was positive for H5N1 avian influenza virus and influenza virus type A. The H5N1 virus strain named A/Zhejiang/16/06 (H5N1) was isolated. The titers of HI antibody for H5N1 avian influenza virus were 1:320 and 1:640, respectively. The sequenced genes were all avian origin. Phylogenetic analyses between the A/Zhejiang/16/06 and other H5N1 influenza viruses were also included.