The threat of highly pathogenic avian influenza (HPAI) H5N1 viruses to cause the next pandemic remains a major concern. Here, we evaluated the cross-protection induced by natural infection of human seasonal influenza strains or immunization with trivalent inactivated influenza vaccine (TIV) against HPAI H5N1 (A/Vietnam/1203/2004) virus in ferrets. Groups were treated with PBS (group A), infected with H1N1 (group B) or H3N2 (group C) virus, or immunized with TIV (group D). Twelve weeks after the last treatment, serological assays revealed that groups B and C, but not group D, sustained moderate immunogenicity against homologous viruses; cross-reactivity against the H5N1 virus was not detected in any group. Following challenge with A/Vietnam/1203/2004 (H5N1) virus, only groups B and C exhibited attenuated viral loads leading to 100 % survival. Our data suggest that natural infection with human seasonal strains could potentially provide better heterosubtypic protection against HPAI H5N1 virus infection compared to TIV immunization.