Vaccination has been an important component of preventative health care programs of North American zoologic institutions in their protection of valuable species against West Nile virus (WNV) infection since its detection in 1999. Although approved only for horses, commercial WNV vaccine has been used for the purpose of protection of nondomestic species, including avian, equid, and rhinoceros species. Currently, there are two commercial equine vaccines available, a killed vaccine and a recombinant viral-vectored vaccine. Both products have been used for the vaccination of Greater One-horned rhinoceroses (Rhinoceros unicornis) held in North American zoologic institutions. In this study, the efficacy of these vaccines was evaluated in Greater One-horned rhinoceroses based on the humoral immune response stimulated by vaccine administration. Five rhinoceroses were vaccinated in 2005 by using the killed equine vaccine and four received boosters in 2006 by using the recombinant vaccine. Rhinoceroses were evaluated for differences in pre- and postvaccination neutralizing antibody titer and gamma and beta globulins on serum protein electrophoresis. No changes were observed after administration of the killed vaccine; however, antibody titers were observed in two of four rhinoceroses after administration of the recombinant vaccine. No significant changes were observed in the serum protein electrophoresis after either vaccine. Based on these findings, the WNV recombinant vaccine appeared to induce a more measurable humoral immune response than the killed product in the Greater One-horned rhinoceros. However, further investigation of both vaccines is warranted to evaluate whether changes in the frequency of administration, dosage, or adjuvant might stimulate an improved humoral response in these animals.