Avian influenza virus is acquiring the ability to cross the species barrier between birds and mammals. However, the genetic mechanism of this ability is not well known. We successfully rescued an influenza A virus A/Chicken/Guangdong/03 (H5N1), which is highly pathogenic to both SPF chickens and BALB/c mice, by plasmid-based reverse genetics. The rescued virus, R-A/Chicken/Guangdong/03 (R-CG), and the wild-type A/Chicken/Guangdong/03 (W-CG) were found to share similar biological properties, such as in titres of 50% egg infectious dose (EID50), 50% tissue culture infectious dose (TCID50) and intravenous pathogenicity index (IPVI). R-CG, like W-CG, is highly pathogenic in mice following natural route infection. Both R-CG and W-CG viruses can be isolated from many organs of mice such as brain, lung, kidney and spleen. As a result, the constructed R-CG reverse genetic system can be used as a tool in the investigation of the molecular mechanism of infection of the mammalian host by avian influenza virus.