We tested the ability of retrocyclin 2, a type of theta defensin, to protect cells and chicken embryos from infection by H5N1 highly pathogenic avian influenza virus. A gene fragment of retrocyclin 2 was designed based on the protein sequence of retrocyclin 2 and cloned into the eukaryotic expression vector pcDNA4.01 (HismaxA), named pcDNA4-RC2. The expression vector pcDNA4-RC2 protected MDCK cells and chicken embryos from infection by the H5N1 virus through inhibition of virus replication and viral mRNA transcription. Retrocyclin 2 is therefore effective in preventing H5N1 virus infection in vivo and in vitro and could be considered as a new therapy for H5N1 influenza and other diseases.