Greater sage-grouse (Centrocercus urophasianus) are an endangered species in Canada and have been extirpated from five states in the United States. Infectious diseases can pose a challenge to conservation efforts, and the greater sage-grouse is susceptible to West Nile virus (WNV). This study measured the humoral response to a commercially available WNV vaccine in a zoo-based conservation breeding population of greater sage-grouse. Since the initiation of the conservation program in 2014, all sage-grouse at the Calgary Zoo's Wildlife Conservation Centre have been vaccinated against WNV. Juveniles received a series of three vaccines every 2–3 wk starting around 2 wk of age; adults had received the same vaccination series at hatch and had since been boostered annually. Antibody titers were measured on 60 serum samples from 36 juveniles and 18 adults collected in 2018 using either serum neutralization or plaque-reduction neutralization methods. No detectable antibody response was noted in juveniles after their second (n = 17) or third booster vaccinations (n = 23). Only 35% of 20 adult samples collected had positive titers even after multiple years of vaccination. Only two cases of adverse vaccine reaction have been noted in this species with over 800 doses being administered between 2014 and 2020. Despite a lack of antibody response in juveniles and most adults, there have been no WNV-associated deaths in this population even with confirmed cases of WNV in free-ranging greater sage-grouse and other species in the area during this time frame, suggesting that cell-mediated immunity may be of greater importance for protection against WNV infections in this species. The initial vaccination series was changed in 2019 to a series of only two doses of vaccine, and no clinical cases or mortality from WNV occurred in 2019 or 2020 with the new protocol.