Beta-defensins are highly conserved cationic peptides strongly involved in mucosal innate defenses of mammals against pathogens, notably bacteria, either by direct antimicrobial effect or indirectly by modulating the host immune responses. In birds, avian beta-defensins (AvBDs) represent the largest family of innate immunity antimicrobial peptides. AvBD2 and AvBD7 are two major beta-defensins found in chicken heterophils and intestinal epithelial cells. We have previously shown that these two AvBDs display broadspectrum antibacterial activity and resistance to proteolysis, thus pointing to a putative important role in mucosa protection in poultry. However, their capacity to control viral infection has yet to be elucidated. Avian influenza viruses (AIV) are worldwide spread and represent a permanent threat to poultry industries, backyard farming and public health. The respiratory and digestive tracts are the principal entry ports for AIV in avian hosts. Therefore, in the present study, we investigated whether AvBDs might limit the replication of an H1N1 subtype avian influenza virus in chicken epithelial cells. Interestingly, AvBD2 unlike AvBD7 was shown to markedly reduce the overall cytotoxic effect of AIV infection in a chicken lung epithelial cell line (CLEC213). These results reveal for the first time substantial differences in the biological activity pattern between avian defensins with AvBD2, but not AvBD7, being able to exert antiviral effects against avian influenza viruses.