Technical Abstract: The negative impact of high pathogenicity avian influenza virus (HPAIV) infection on egg production and deposition of virus in eggs, as well as any protective effect of vaccination, is unknown. Individually housed non-vaccinated, sham-vaccinated and inactivated H5N9 vaccinated once or twice adult White leghorn hens were challenged intranasally/intratracheally 3-weeks post-vaccination with H5N2 HPAIV. The non-/sham-vaccinated layers experienced 100% mortality (0% survivability) within 3-4 days post-challenge (DPC), and major changes to reproductive parameters including precipitous drops in egg production (79% to 0% in < 5 days), production of soft and thin-shelled eggs, and deposition of virus in albumin and yolk, and on the egg shell surface of 53% of eggs. By comparison, the three H5-vaccinated groups had 83%, 100% and 100% survivability after challenge; The two H5-vaccinated once hens that died had low pre-challenge HI titers (GMT=16). H5-vaccinated once or twice groups maintained egg production after challenge (63%), but there was a mild and significant reduction in egg production as compared to pre-challenge egg production (79%). H5-vaccinated groups had reduced number of virus contaminated eggs (28%), and in most groups, reduced quantity of virus in contaminated eggs compared to non-/sham-vaccinated groups. No HPAIV-positive eggs were laid on or after 5 DPC. In conclusion, HPAIV infection had major negative impact on egg production and other reproductive parameters. H5-vaccination once or twice prevented declines in egg production after HPAIV challenge, reduced number of virus-infected eggs, and usually reduced the titer of virus in internal contents and on eggshell surface, suggesting that protection from HPAIV is more difficult in reproductively active chickens than in growing young chickens.