The loggerhead shrike (Lanius ludovicianus migrans) is a migratory songbird that has undergone massive population declines in Ontario since the 1950s. As part of a broad strategy of recovery, a captive breeding population was established in the late 1990s. This species appears to be extremely sensitive to West Nile virus (WNV) infection, with prior outbreaks at Ontario breeding facilities reaching a 100% mortality rate. This study aimed to investigate the humoral response to vaccination in juvenile birds given single versus serial booster vaccinations, as well as to assess the duration of protective virus-neutralizing titers in annually vaccinated adult birds, by measuring WNV-neutralizing antibodies via the Plaque Reduction Neutralization Test. Twenty-two adult birds and forty 18–22-day-old chicks were included in the study. Annual vaccination resulted in serum neutralizing antibody against WNV for only 59% of adult individuals 1 yr following vaccination. These results, coupled with the death of one vaccinated adult individual due to WNV infection, suggest that a second booster vaccination may be required to adequately protect adult individuals throughout the WNV transmission season. The results of the trial involving juvenile birds indicate that vaccination does not effectively stimulate the immune system of naïve juveniles to produce serum-neutralizing antibodies against WNV in the majority of tested birds, although serial booster vaccination appears to provide a level of improved seroconversion. However, the loss of 19% of naïve juveniles to natural WNV infection versus a less than 3% loss of juveniles that received at least one vaccination suggests some level of cell-mediated immunity and protection against infection takes place in juvenile birds postvaccination. The deaths of several nonvaccinated juveniles and one vaccinated adult at this study facility suggest that WNV continues to be a pathogen of high risk in this species in captivity, and likely in the wild as well.