Description: Objective--To evaluate the long-term protective immunity of a cyprinid herpesvirus 3 (CyHV3) vaccine in naive koi (Cyprinus carpio koi). Animals--72 koi. Procedures--Vaccinated koi (n = 36) and unvaccinated control koi (36) were challenge exposed to a wild-type CyHV3 strain (KHVp8 F98-50) 13 months after vaccination. Results--The CyHV3 vaccine provided substantial protective immunity against challenge exposure. The proportional mortality rate was less in vaccinated koi (13/36 [36 percent ) than in unvaccinated koi (36/36 [100 percent) For koi that died during the experiment, mean survival time was significantly greater in vaccinated than in unvaccinated fish (17 vs 10 days). Conclusions and Clinical Relevance--The CyFIV3 vaccine provided substantial protective immunity against challenge exposure with CyHV3 13 months after vaccination. This provided evidence that koi can be vaccinated annually with the CyFIV3 vaccine to significantly reduce mortality and morbidity rates associated with CyFIV3 infection